Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).

The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).

The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).

Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).

It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).

It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).

It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).

The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSX^M is required to promote male development and repress female development, while the opposite is true for DSX^F. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSX^M in promoting embryonic hub formation, while DSX^F is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).

We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSX^F to repress male development in females. However, DSX^M may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).

The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).

In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).

In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

One consequence of this work is to lead to a reevaluation of the differences between male and female gonad development in Drosophila. Indeed, Hh signaling is an essential regulator of the self-renewal and the number of follicle stem cells, the offspring of which carry out a comparable function to cyst cells by ensheathing germ line cysts. In the ovary, as in the testis, JAK/STAT signaling in somatic cells is required for the maintenance of GSCs via BMP production. However, in the ovary, the escort cells and cap cells are the JAK/STAT-responsive niche cells, implying that CySCs in the male gonad fulfill the function of two cell types in the female gonad and require both the signals used in the female to do so. Finally, the data evoke the interesting possibility that Hh has a conserved ancestral role in male gonads. Mutation in one of the three mammalian hh homologs, desert hedgehog (Dhh), causes male sterility and a loss of somatic support cells called Leydig cells. However, the cellular niche for spermatogenesis in mammals is less well understood than in Drosophila and it remains to be established whether the Hh pathway orchestrates similar cellular functions (Amoyel, 2013).

Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. This study shows that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. The study proposes that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling (Inaba, 2015).

The Drosophila testis represents an excellent model system to study niche-stem-cell interactions because of its well-defined anatomy: eight to ten germline stem cells (GSCs) are attached to a cluster of somatic hub cells, which serve as a major component of the stem cell niche. The hub secretes at least two ligands: the cytokine-like ligand Unpaired (Upd), and a BMP ligand Decapentaplegic (Dpp), both of which regulate GSC maintenance. GSCs typically divide asymmetrically, so that one daughter of the stem cell division remains attached to the hub and retains stem cell identity, while the other daughter, called a gonialblast, is displaced away from the hub and initiates differentiatio. Given the close proximity of GSCs and gonialblasts, the ligands (Upd and Dpp) must act over a short range so that signalling is only active in stem cells, but not in differentiating germ cells. The basis for this sharp boundary of pathway activation remains poorly understood (Inaba, 2015).

Using green fluorescent protein (GFP)-α1-tubulin84B expressed in germ cells (nos-gal4>UAS-GFP-αtub), this study found that GSCs form protrusions, referred to as microtubule-based (MT)-nanotubes hereafter, that extend into the hub. MT-nanotubes are sensitive to fixation similar to other thin protrusions reported so far, such as tunnelling nanotubes, explaining why they have escaped detection in previous studies. MT-nanotubes appear to be specific to GSCs: 6.67 MT-nanotubes were observed per testis in the GSC population (or 0.82 per cell). The average thickness and length of MT-nanotubes are 0.43 ± 0.29 µm (at the base of MT-nanotube) and 3.32 ± 1.6 µm, respectively. These GSC MT-nanotubes are uniformly oriented towards the hub area. By contrast, differentiating germ cells showed only 0.44 MT-nanotubes per testis (or <0.002 per cell), without any particular orientation when present. MT-nanotubes were sensitive to colcemid, the microtubule-depolymerizing drug, but not to the actin polymerization inhibitor cytochalasin B, suggesting that MT-nanotubes are microtubule-based structures. MT-nanotubes were not observed in mitotic GSCs, and GSCs form new MT-nanotubes as they exit from mitosis. By contrast, MT-nanotubes in interphase GSCs were stably maintained for up to 1 h of time-lapse live imaging. Although cell-cycle-dependent formation of MT-nanotube resembles that of primary cilia, MT-nanotubes are distinct structures, in that they lack acetylated microtubules and are sensitive to fixation. Furthermore, a considerable fraction of GSCs form multiple MT-nanotubes per cell (54% of GSCs with MT-nanotubes), and MT-nanotubes are not always associated with the centrosome/basal body, as is the case for the primary cilia (Inaba, 2015).

To examine the geometric relationship between MT-nanotubes and hub cells further, MT-nanotubes were imaged in combination with various cell membrane markers, followed by three-dimensional rendering. Although the MT-nanotubes are best visualized in unfixed testes that express GFP-αTub in germ cells, adding a low concentration (1 μM) of taxol to the fixative preserves MT-nanotubes, allowing immunofluorescence staining. First, Armadillo (Arm, β-catenin) staining, which marks adherens junctions formed at hub cell/hub cell as well as hub cell/GSC boundaries, revealed that adherens junctions do not form on the surface of MT-nanotubes. Using FM4-64 styryl dye, it was found that the MT-nanotubes are ensheathed by membrane lipids. Furthermore, myristoylation/palmitoylation site GFP (myrGFP), a membrane marker, expressed in either the germline or hub cells illuminated MT-nanotubes, suggesting that the surface membrane of a MT-nanotube is juxtaposed to hub-cell plasma membrane (Inaba, 2015).

Genes were examined that regulate primary cilia and cytonemes for their possible involvement in MT-nanotube formation. RNA interference (RNAi)-mediated knockdown of oseg2 (IFT172), osm6 (IFT52) and che-13 (IFT57), components of the intraflagellar transport (IFT)-B complex that are required for primary cilium anterograde transport and assembly, significantly reduced the length and the frequency of MT-nanotubes. Knockdown of Dlic, a dynein intermediate chain required for retrograde transport in primary cilia<, also reduced the MT-nanotube length and frequency. Knockdown of klp10A, a Drosophila homologue of mammalian kif24 (a MT-depolymerizing kinesin of the kinesin-13 family, which suppresses precocious cilia formation), resulted in abnormally thick/bulged MT-nanotubes. No significant changes were observed in MT-nanotube morphology upon knockdown of IFT-A retrograde transport genes, such as oseg1 and oseg3 (Inaba, 2015).

Endogenous Klp10A localized to MT-nanotubes both in wild-type testes and in GFP-αTub-expressing testes. GFP-Oseg2 (IFT-B), GFP-Oseg1, GFP-Oseg3 (IFT-A) and Dlic also localized to the MT-nanotubes when expressed in germ cells. The localization of IFT-A components to MT-nanotubes, without detectable morphological abnormality upon mutation/knockdown, is reminiscent of the observation that most of the genes for IFT-A are not required for primary cilia assembly. Expression of a dominant negative form of Dia (Dia^DN) or a temperature-sensitive form of Shi (Shi^ts) in germ cells (nos-gal4>UAS-dia^DN or UAS-shi^ts), which perturb cytoneme formation, did not influence the morphology or frequency of MT-nanotubes in GSCs. Taken together, these results show that primary cilia proteins localize to MT-nanotubes and regulate their formation (Inaba, 2015).

In search of the possible involvement of MT-nanotubes in hub-GSC signalling, it was found that the Dpp receptor, Thickveins (Tkv), expressed in germ cells (nos-gal4>tkv-GFP) was observed within the hub region, in contrast to GFP alone, which remained within the germ cells. A GFP protein trap of Tkv (in which GFP tags Tkv at the endogenous locus) also showed the same localization pattern as Tkv-GFP expressed by nos-gal4. By inducing GSC clones that co-express Tkv-mCherry and GFP-αTub, it was found that Tkv-mCherry localizes along the MT-nanotubes as puncta. Furthermore, using live observation, Tkv-mCherry puncta were observed to move along the MT-nanotubes marked with GFP-αTub, suggesting that Tkv is transported towards the hub along the MT-nanotubes. It should be noted that, in the course of this study, it was noticed that mCherry itself localized to the hub when expressed in germ cells, similar to Tkv-GFP and Tkv-mCherry. Importantly, the receptor for Upd, Domeless (Dome), predominantly stayed in the cell body of GSCs, demonstrating the specificity/selectivity of MT-nanotubes in trafficking specific components of the niche signalling pathways. A reporter of ligand-bound Tkv, TIPF localized to the hub region together with Tkv-mCherry, in addition to its reported localization at the hub-GSC interface. Furthermore, Dpp-GFP expressed by hub cells co-localized with Tkv-mCherry expressed in germline. These results suggest that ligand (Dpp)-receptor (Tkv) engagement and activation occurs at the interface of the MT-nanotube surface and the hub cell plasma membrane. Knockdown of IFT-B components (oseg2^RNAi, che-13^RNAi or osm6^RNAi), which reduces MT-nanotube formation, resulted in reduction of the number of Tkv-GFP puncta in the hub area, concomitant with increased membrane localization of Tkv-GFP. A similar trend was observed upon treatment of the testes with colcemid, suggesting that MT-nanotubes are required for trafficking of Tkv into the hub area. By contrast, knockdown of Klp10A, which causes thickening of MT-nanotubes, led to an increase in the number of Tkv-GFP puncta in the hub area. Taken together, these data suggest that Tkv is trafficked into the hub via MT-nanotubes, where it interacts with Dpp secreted from the hub (Inaba, 2015).

Knockdown of klp10A (klp10A^RNAi) led to elevated phosphorylated Mad (pMad) levels, a readout of Dpp pathway activation, in GSCs. By contrast, RNAi-mediated knockdown of oseg2, osm6 and che-13 (IFT-B components), which causes shortening of MT-nanotubes, reduced the levels of pMad in GSCs. Dad-LacZ, another readout of Dpp signalling activation, exhibited clear upregulation upon knockdown of klp10A. GSC clones of che-13^RNAi, osm6^RNAi or oseg2⁴⁵² were lost rapidly compared with control clones, consistent with the idea that MT-nanotubes help to promote Dpp signal transduction. Knockdown of oseg2, che-13 and osm6 did not visibly affect cytoplasmic microtubules, suggesting that GSC maintenance defects upon knockdown of these genes are probably mediated by their role in MT-nanotube formation. Global RNAi knockdown of these genes in all GSCs using nos-gal4 did not cause a significant decrease in GSC numbers , indicating that compromised Dpp signalling due to MT-nanotube reduction leads to a competitive disadvantage in regards to GSC maintenance only when surrounded by wild-type GSCs (Inaba, 2015).

When klp10A^RNAi GSC clones were induced, pMad levels specifically increased in those GSC clones, indicating that Klp10A acts cell-autonomously in GSCs to influence Dpp signal transduction. Importantly, klp10A^RNAi spermatogonia did not show a significant elevation in pMad level compared with control spermatogonia, demonstrating that the role of Klp10A in regulation of Dpp pathway is specific to GSCs. pMad levels did not change in spermatogonia upon manipulation of MT-nanotube formation. GSC clones of klp10A^RNAi or klp10A null mutant (klp10A²⁴) did not dominate in the niche, despite upregulation of pMad, possibly because of its known role in mitosis. Importantly, these conditions did not significantly change STAT92E levels, which reflect Upd-JAK-STAT signalling in GSCs, revealing the selective requirement of MT-nanotubes in Dpp signalling. Together, these results demonstrate that MT-nanotubes specifically promote Dpp signalling and their role in enhancing the Dpp pathway is GSC specific (Inaba, 2015).

Since cytonemes are induced/stabilized by the signalling molecules themselves, the possible involvement of Dpp in MT-nanotube formation was explored First, it was found that a temperature-sensitive dpp mutant (dpp^hr56/dpp^hr4) exhibited a dramatic decrease in the frequency of MT-nanotubes (0.067 MT-nanotubes per GSC) and the remaining MT-nanotubes were significantly thinner. Knockdown of tkv (tkv^RNAi) in GSCs also resulted in reduced length and frequency of MT-nanotubes. Conversely, overexpression of Tkv (tkv^OE) in germ cells led to significantly longer MT-nanotubes. Interestingly, expression of a dominant negative Tkv (tkv^DN), which has intact ligand-binding domain but lacks its intracellular GS domain and kinase domain, resulted in thickening of MT-nanotubes, rather than reducing the thickness/length. This indicates that ligand-receptor interaction, but not downstream signalling events, is sufficient to induce MT-nanotube formation. Strikingly, upon ectopic expression of Dpp in somatic cyst cells (tj-lexA>dpp), spermatogonia/spermatocytes were observed to have numerous MT-nanotubes, suggesting that Dpp is necessary and sufficient to induce or stabilize MT-nanotubes in the neighbouring germ cells. In turn, MT-nanotubes may promote selective ligand-receptor interaction between hub and GSCs, leading to spatially confined self-renewal (Inaba, 2015).

This study shows that previously unrecognized structures, MT-nanotubes, extend into the hub to mediate Dpp signalling. It is proposed that MT-nanotubes form a specialized cell surface area, where productive ligand-receptor interaction occurs. In this manner, only GSCs can access the source of highest ligand concentration in the niche via MT-nanotubes, whereas gonialblasts do not experience the threshold of signal transduction necessary for self-renewal, contributing to the short-range nature of niche signalling. In summary, the results reported here illuminate a novel mechanism by which the niche specifies stem cell identity in a highly selective manner (Inaba, 2015).

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. This study shows that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. The driving force behind such behavior is accelerated proliferation. These results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process (Amoyel, 2014).

Finally, the fact that lines-depleted CySCs recruited neighboring wild-type somatic cells to adopt CySC fate is striking. Although the imaging tools necessary to reveal which somatic cells are recruited to CySC fate are unavailable, the fact of their recruitment suggests that under these mutant conditions cyst cells can de-differentiate into CySCs. It has been elegantly shown that maturing germ cells can de-differentiate, creating new GSCs. As those maturing germ cells are encysted by the somatic cyst cells, during de-differentiation this grouping must break apart to release individual germline cells that repopulate the niche. Whether cyst cells de-differentiate to CySCs in these cases has not been directly assessed. If this happens under physiological conditions, it would be of great interest to study how cyst cells de-differentiation occurs, and testes harboring lines-deficient clones might aid in such studies (Dinardo, 2011).

Drosophila is widely used to analyse functions of different genes. The phosphatidylcholine lysophospholipase gene swiss cheese was initially shown to be important in the fruit fly nervous system. However, the role of this gene in non-nervous cell types has not been elucidated yet, and the evolutional explanation for the conservation of its function remains elusive. This study analyses the expression pattern and some aspects of the role of the swiss cheese gene in the fitness of Drosophila melanogaster. The spatiotemporal expression of swiss cheese throughout the fly development is described and the survival and productivity of swiss cheese mutants were analyzed. swiss cheese was found to be expressed in salivary glands, midgut, Malpighian tubes, adipocytes, and male reproductive system. Dysfunction of swiss cheese results in severe pupae and imago lethality and decline of fertility, which is impressive in males. The latter is accompanied with abnormalities of male locomotor activity and courtship behavior, accumulation of lipid droplets in testis cyst cells and decrease in spermatozoa motility. These results suggest that normal swiss cheese is important for Drosophila melanogaster fitness due to its necessity for both specimen survival and their reproductive success (Melentev, 2021).

Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w¹¹¹⁸ progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

Tight junctions prevent paracellular flow and maintain cell polarity in an epithelium. These junctions are also required for maintaining the blood-testis-barrier, which is essential for sperm differentiation. Septate junctions in insects are orthologous to the tight junctions. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. Characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with previous reports, knockdown of essential junctional components- Discs-large-1 and Neurexin-IV- during the early stages disrupted sperm differentiation beyond the spermatocyte stage. Knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining the integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during mammalian spermatogenesis (Dubey, 2019).

Spermatogenesis is a dynamic developmental process requiring precisely timed transitions between discrete stages. Specifically, the germline undergoes three transitions: from mitotic spermatogonia to spermatocytes, from meiotic spermatocytes to spermatids, and from morphogenetic spermatids to spermatozoa. The somatic cells of the testis provide essential support to the germline throughout spermatogenesis, but their precise role during these developmental transitions has not been comprehensively explored. This study describes the identification and characterization of genes that are required in the somatic cells of the Drosophila melanogaster testis for progress through spermatogenesis. Phenotypic analysis of candidate genes pinpointed the stage of germline development disrupted. Bioinformatic analysis revealed that particular gene classes were associated with specific developmental transitions. Requirement for genes associated with endocytosis, cell polarity, and microtubule-based transport corresponded with the development of spermatogonia, spermatocytes, and spermatids, respectively. Overall, this study identified mechanisms that act specifically in the somatic cells of the testis to regulate spermatogenesis (Fairchild, 2017).

The stem cell niche normally prevents aberrant stem cell behaviors that lead to cancer formation. Recent studies suggest that some cancers are derived from endogenous populations of adult stem cells that have somehow escaped from normal control by the niche. However, the molecular mechanisms by which the niche retains stem cells locally and tightly controls their divisions are poorly understood. This study demonstrates that the presence of heparan sulfate (HS), a class glygosaminoglycan chains, in the Drosophila germline stem cell niche prevents tumor formation in the testis. Loss of HS in the niche, called the hub, led to gross changes in the morphology of testes as well as the formation of both somatic and germline tumors. This loss of hub HS resulted in ectopic signaling events in the Jak/Stat pathway outside the niche. This ectopic Jak/Stat signaling disrupted normal somatic cell differentiation, leading to the formation of tumors. This finding indicates a novel non-autonomous role for niche HS in ensuring the integrity of the niche and preventing tumor formation (Levings, 2017).

Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). This study found that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor Doublesex^M (Dsx^M) within CySCs and their progeny, and ectopic expression of Dsx^M in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of Dsx^M. The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. This work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals (Ma, 2014).

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, this study showed that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. It is proposed that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells (Qian, 2014).

Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. This study shows that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, TFIIH stability was not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients (Cruz-Becerra, 2016).

Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. This study reports that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. The results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. dUTX acts in cyst stem cells (CySCs) to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, these findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo (Tarayrah, 2013).

This study identified a new epigenetic mechanism that negatively regulates the JAK-STAT signaling pathway in the Drosophila testis niche: the H3K27me3-specific demethylase dUTX acts in CySCs to remove the repressive H3K27me3 histone modification near the TSS of Socs36E to allow its active transcription. Socs36E acts upstream to suppress Stat92E activity and to restrict CySCs from overpopulating the testis niche. In addition, dUTX acts in CySCs to prevent hyperactivation of Stat92E in hub cells, which would otherwise ectopically turn on Zfh1 expression. When zfh1 cDNA was ectopically driven in hub cells using the upd-Gal4 driver, no obvious defect could be identified. Therefore, the biological consequence of ectopic Zfh1 expression in hub cells remains unclear. However, ectopic Zfh1 expression in hub cells and the overpopulation of Zfh1-expressing cells around the hub are two connected phenomena because both phenotypes are caused by loss of dUTX in CySCs (Tarayrah, 2013).

UTX also acts in GSCs to regulate DE-Cadherin levels to maintain proper GSC-hub interaction and normal morphology of the hub. It has been reported that differential expression of different cadherins causes cells with similar cadherin types and levels to aggregate. In wt testes, hub cells express higher levels of DE-Cadherin and therefore tightly associate with each other. Loss of dUTX in germ cells leads to higher levels of DE-Cadherin in GSCs, which probably allows them to intermingle with hub cells and causes disrupted hub architecture. It has also been demonstrated that the major role of JAK-STAT in GSCs is for GSC-hub adhesion, suggesting that the expression and/or activity of cell-cell adhesion molecules, such as DE-Cadherin, depends on JAK-STAT signaling. Therefore, the abnormal DE-Cadherin activity in GSCs in dUTX testis could also result from misregulated JAK-STAT signaling in the testis niche. dUTX is a new negative epigenetic regulator of the JAK-STAT signaling pathway (Tarayrah, 2013).

The JAK-STAT signaling pathway plays crucial roles in stem cell maintenance in many different stem cell types across a wide range of species. These studies identify the histone demethylase dUTX as a new upstream regulator of the JAK-STAT pathway, which directly controls the transcription of Socs36E. In addition to acting as an antagonist of JAK-STAT signaling, Socs36E has been reported to be a direct target gene of the Stat92E transcription factor (Terry, 2006). Therefore, increased Stat92E would be expected to upregulate Socs36E expression, but this was not observed in dUTX mutant testes. Instead, the data revealed that Socs36E expression decreased, whereas Stat92E expression increased, in dUTX testes, consistent with the hypothesis that Socs36E is a direct target gene of dUTX and acts upstream of Stat92E (Tarayrah, 2013).

Sustained activity of the JAK-STAT pathway in cyst cells has been reported to activate BMP signaling, which leads to GSC self-renewal outside the niche and gives rise to a tumor-like phenotype in testis. To examine BMP pathway activity, immunostaining experiments were performed using antibodies against phospho-SMAD (pSMAD), a downstream target of BMP signaling. No obvious difference was detected in the pSMAD signal between the dUTX testes and wt control, nor were any germline tumors detected in dUTX testes. It is speculated that germline tumor formation upon activation of the JAK-STAT pathway is secondary to the overproliferation of Zfh1-expressing cells, which was not observed in dUTX testes (Tarayrah, 2013).

This study also provides an example of the multidimensional cell-cell communication that takes place within a stem cell niche. Many studies of the stem cell niche have focused on understanding niche-to- stem cell signaling in controlling stem cell identity and activity. For example, in the Drosophila female GSC niche, Upd secreted from terminal filaments activates the JAK-STAT pathway in cap cells and escort cells, which subsequently produce the BMP pathway ligand Decapentaplegic (Dpp) to activate BMP signaling and prevent transcription of the differentiation factor bag-of-marbles (bam) in GSCs. In the Drosophila intestinal stem cell (ISC) niche, the visceral muscle cells underlying the intestine secrete Wingless to activate Wnt signaling and Upd to activate JAK-STAT signaling in ISCs, which are required to maintain ISC identity and activity (Tarayrah, 2013).

More studies have now revealed the multidirectionality of signaling within the stem cell niche. For example, in the Drosophila female GSC niche, GSCs activate Epidermal growth factor receptor (Egfr) signaling in the neighboring somatic cells, which subsequently represses expression of the glypican Dally, a protein required for the stabilization and mobilization of the BMP pathway ligand Dpp. Through this communication between GSCs and the surrounding somatic cells, only GSCs maintain high BMP signaling. The current studies establish another example of the multidimensional cell-cell communications that occur within the testis stem cell niche, where CySCs and GSCs have distinct roles in regulating hub cell identity and morphology (Tarayrah, 2013).

The data identified new roles of a histone demethylase in regulating endogenous stem cell niche architecture and proper gene expression. Previous studies have reported in vivo functions of histone demethylases in several model organisms. For example, mammalian UTX has been shown to associate with the H3K4me3 histone methyltransferase MLL2, suggesting its potential antagonistic role to the PcG proteins. The PcG proteins play a crucial role in Hox gene silencing in both Drosophila and mammals. Consistently, mammalian UTX has been reported to directly bind and activate the HOXB1 gene locus. In addition to antagonizing PcG function, H3K27me3 demethylases play crucial roles during development. For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development, and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development, including gonad development. Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation, and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency. These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming. However, despite multiple reports on the in vivo roles of H3K27me3-specific demethylases, little is known about their functions in any endogenous adult stem cell system (Tarayrah, 2013).

Whereas mammals have multiple H3K27me3 demethylases, dUTX is the sole H3K27me3-specific demethylase in Drosophila. This unique feature, plus the well-characterized nature of Drosophila adult stem cell systems, make interpretation of the endogenous functions of histone demethylases in Drosophila unambiguous. Because mammalian UTX has been reported as a tumor suppressor, understanding the endogenous functions of dUTX in an adult stem cell system might facilitate the use of histone demethylases for cancer treatment. In summary, these results demonstrate that stem cells send feedback to the niche cells to maintain their proper gene expression and morphology. Furthermore, this feedback is regulated through the JAK-STAT signaling pathway, the activity of which is controlled by a chromatin factor, providing an example of crosstalk between these two regulatory pathways (Tarayrah, 2013).

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). This study shows a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. This work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche (Stine, 2014: PubMed).

Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, is the first identified regulator of niche competition in the Drosophila testis. The competitive behavior of (Suppressor of cytokine signaling at 36E) (Socs36E) mutant cyst stem cells (CySCs) has been attributed to increased JAK/STAT signaling. This study shows that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, it was found that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of germ line stem cells, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, these results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is the node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior (Amoyel, 2016a).

Stem cell niches are complex environments that provide support for stem cells through molecular signals. Several well-characterized niches provide not just one but multiple signals which stem cells must integrate and interpret in order to remain at the niche and self-renew. How this integration is achieved is not well understood at present. Furthermore, in order to maintain the appropriate number of stem cells and the homeostatic balance between self-renewal and differentiation, it is necessary that self-renewal cues be present in limiting amounts or that their activity be dampened to prevent excessive accumulation of stem cells. One general feature of many signal transduction pathways is the presence of feedback inhibitors. These are dampeners of signaling, transcriptionally induced by the signaling itself, that prevent signal levels from being aberrantly high. One such family of feedback inhibitors is the Suppressor of Cytokine Signaling (SOCS) proteins, which were identified as inhibitors of JAK/STAT signal transduction, and are SH2- and E3-ligase domain-containing proteins. The SH2 domain binds phosphorylated (i.e., activated) signal transduction components and the E3-ligase targets them for degradation by Ubiquitin-dependent proteolysis. In mammals, SOCS proteins can thus inhibit several tyrosine kinase-dependent signaling pathways, including JAK/STAT and Mitogen-Activated Protein Kinase (MAPK) (Amoyel, 2016a).

The Drosophila testis is an ideal model system to study questions of signal regulation and integration in stem cells. The testis niche, called the hub, supports two stem cell populations. The first, germ line stem cells (GSCs), gives rise to sperm after several transit-amplifying divisions leading up to meiosis. The second, somatic cyst stem cells (CySCs), gives rise to cyst cells, the essential support cells for germ line development. Many ligands for signaling pathways are produced by the hub, including the JAK/STAT pathway agonist, Unpaired (Upd), the Hedgehog (Hh) pathway ligand Hh and the Bone Morphogenetic Protein (BMP) homologs Decapentaplegic (Dpp) and Glass Bottom Boat (Gbb). The latter two signals are also produced by CySCs and are required in GSCs for self-renewal, indicating that CySCs constitute part of the niche for GSCs along with the hub. CySCs require JAK/STAT and Hh activity for self-renewal (Amoyel, 2016a).

CySCs and GSCs compete for space at the niche, a phenomenon that was revealed by the analysis of testes lacking the JAK/STAT feedback inhibitor Socs36E. In these animals, excessive JAK/STAT activity was detected in CySCs, and Socs36E mutant CySCs displaced the resident wild type GSCs. Additionally, it has been shown that CySCs with sustained Hh signaling or sustained Yorkie (Yki) activity also outcompeted neighboring wild type GSCs, indicating that several signaling pathways can control niche competition. Moreover, prior to out-competing GSCs, mutant CySCs displaced neighboring wild type CySCs, indicating that both intra- (CySC-CySC) and inter-lineage (CySC-GSC) competition take place in the testis. While the two types of competition appear related, in that one precedes the other, there are instances in which only intra-lineage competition takes place. While the competitive phenotype of Socs36E mutant CySCs was ascribed to increased JAK/STAT signaling, it was surprising to find that clonal gain-of-function in JAK/STAT signaling in CySCs did not induce competitive behavior, and it was concluded that loss of Socs36E did not mimic increased JAK/STAT signaling in CySC (Amoyel, 2016a).

This study addressed whether other mechanisms could account for the competitive behavior of Socs36E mutant CySCs. Because SOCS proteins can inhibit MAPK signaling in cultured cells and in Drosophila epithelial tissues, this study examined if Socs36E repression of MAPK signaling underlied the Socs36E competitive phenotype. Indeed, it was found that Socs36E inhibits MAPK signaling in CySCs during self-renewal, and that gain of MAPK activity induces CySCs to outcompete wild type CySCs and GSCs at the niche. This study dissected the genetic relationship between Socs36E and the MAPK and JAK/STAT pathways and shows that loss of Socs36E can compensate for decreased self-renewal signaling within CySCs. Thus, CySCs integrate multiple self-renewal signals through the use of a feedback inhibitor that controls at least two signaling pathways regulating stem cell maintenance at the niche (Amoyel, 2016a).

The data presented in this study implicate MAPK signaling as a major regulator of CySC competition for niche access and establish that the competitiveness of CySCs lacking Socs36E is derived primarily from their increased MAPK activity. The ability of a stem cell to self-renew reflects not only intrinsic properties but also extrinsic relationships with its neighbors. For instance, if a cell is unable to compete for space at the niche then it will be no longer able to receive short-range niche signals and will be more likely to differentiate. Conversely, if a cell is more competitive for niche space, this cell and its offspring will replace wild type neighbors and colonize the entire niche. (Amoyel, 2016a).

These data show that CySCs with increased MAPK signaling out-compete neighboring stem cells in CySC-CySC as well as CySC-GSC competition and that CySCs with reduced MAPK activity are themselves out-competed. The interpretation is favored that MAPK regulates primarily competitiveness rather than self-renewal because while MAPK mutant clones are lost from the niche, lineage-wide inhibition of the pathway does not result in a complete loss of stem cells. This contrasts with the role of JAK/STAT signaling in CySCs. Stat92E mutant CySCs are lost and lineage-wide pathway inhibition results in pronounced and rapid stem cell loss. Based on these results, it is argued that JAK/STAT signaling in CySCs primarily controls their intrinsic self-renewal capability while MAPK signaling regulates their competitiveness. Interestingly, there are important similarities between Hh and MAPK function in CySCs in that CySCs lacking Hh signal transduction are out-competed and those with sustained Hh activity out-compete wild type neighbors. Lastly, it is noted that CySCs mutant for the tumor suppressor Hippo (Hpo) (which leads to sustained Yki activation) or Abelson kinase (Abl) also have increased competitiveness, suggesting the existence of multiple inputs controlling the ability of stem cells to stay in the niche at the expense of their neighbors. In the future, it would be interesting to determine if genetic hierarchies exist between competitive pathways or if they independently converge on similar targets. One outstanding question is how altering the competitiveness of CySCs affects the maintenance of the germ line. In the case of Socs36E, MAPK, Hh and Hpo, the competitive CySC displaces not only wild type CySCs but also wild type GSCs. While these observations suggest that out-competition of CySCs and GSCs is linked, the result that Abl mutant CySCs only compete with CySCs and not with GSCs indicates that these two competitive processes are separable genetically (Amoyel, 2016a).

It is well established that Egfr/MAPK signaling is required in somatic cells for their proper differentiation and for their encystment of the developing germ line. In this study, an additional function for Egfr/MAPK was identified in the somatic stem cells, specifically that this pathway regulates competitiveness of CySCs, with each other and with GSCs. Regarding the latter, it is possible that the loss of GSCs when somatic cells have high MAPK signaling is linked to their possibly increased encystment by these cells. Indeed, recent work has shown that Egfr activity in CySCs regulates cytokinesis and maintenance stem cell fate in GSCs. It is tempting to speculate that increased somatic Egfr activity leads to increased encystment of GSCs and loss of stem cell fate in GSCs (Amoyel, 2016a).

MAPK may play a conserved role in niche competitiveness as mouse intestinal stem cells that acquire activating mutations in Ras bias normal stem cell replacement dynamics and colonize the niche. Interestingly, the activating ligand Spi is produced by germ cells, suggesting that the germ line coordinates multiple behaviors in the somatic cell lineage. In addition to transducing signals from the germ line, CySCs also receive ligands from hub cells (including Hh and the JAK/STAT ligand Upd) and they have to integrate these various stimuli. If unmitigated, the combined effect of all of these signals could produce highly competitive CySCs, with overall negative effects on niche homeostasis. The data are consistent with a model in which the induction of Socs36E by the primary self-renewal pathway (JAK/STAT) results in the restraint of a competitive trigger (MAPK) in CySCs. In this way, Socs36E acts to integrate signals from different sources and maintain homeostatic balance between resident cell populations that share a common niche (Amoyel, 2016a).

Stem cell competition has emerged as a mechanism for selecting fit stem cells/progenitors and controlling tumourigenesis. However, little is known about the underlying molecular mechanism. This study identified Mlf1-adaptor molecule (Madm), a novel tumour suppressor that regulates the competition between germline stem cells (GSCs) and somatic cyst stem cells (CySCs) for niche occupancy. Madm knockdown results in overexpression of the EGF receptor ligand vein (vn), which further activates EGF receptor signalling and integrin expression non-cell autonomously in CySCs to promote their overproliferation and ability to outcompete GSCs for niche occupancy. Conversely, expressing a constitutively activated form of the Drosophila JAK kinase (hop(Tum-l)) promotes Madm nuclear translocation, and suppresses vn and integrin expression in CySCs that allows GSCs to outcompete CySCs for niche occupancy and promotes GSC tumour formation. Tumour suppressor-mediated stem cell competition presented in this study could be a mechanism of tumour initiation in mammals (Singh, 2016).

Cdc14 is an evolutionarily conserved serine/threonine phosphatase. Originally identified in S. cerevisiae as a cell cycle regulator, its role in other eukaryotic organisms remains unclear. In Drosophila melanogaster, Cdc14 is encoded by a single gene, thus facilitating its study. Cdc14 expression is highest in the testis of adult flies and cdc14 null flies are viable. cdc14 null female and male flies do not display altered fertility. cdc14 null males, however, exhibit decreased sperm competitiveness. Previous studies have shown that Cdc14 plays a role in ciliogenesis during zebrafish development. In Drosophila, sensory neurons are ciliated. The Drosophila cdc14 null mutants have defects in chemosensation and mechanosensation as indicated by decreased avoidance of repellant substances and decreased response to touch. In addition, it was shown that cdc14 null mutants have defects in lipid metabolism and resistance to starvation. These studies highlight the diversity of Cdc14 function in eukaryotes despite its structural conservation (Neitzel, 2018).

Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. This study performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. The data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. Two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3, were also characterized. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis (Theofel, 2014).

Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). This study identified LADs and TADs at two stages of Drosophila spermatogenesis - in bam^Δ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). This study found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. It was also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, the specific X chromosome organization was uncovered in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG (Ilyin, 2022).

To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. This study shows in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg; CG5204), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. These results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation (Kim, 2017).

Highly specialized cell types such as red blood cells, intestinal epithelium, and spermatozoa are produced throughout life from adult stem cells. In such lineages, mitotically dividing precursors commonly stop proliferation and initiate a cell type-specific transcription program that sets up terminal differentiation of the specialized cell type. In the Drosophila male germ line, stem cells at the apical tip of the testis self-renew and produce daughter cells that each undergo four rounds of spermatogonial mitotic transit amplifying (TA) divisions, after which the germ cells execute a final round of DNA synthesis (premeiotic S-phase) and initiate terminal differentiation as spermatocytes. Transition to the spermatocyte state is accompanied by transcriptional activation of more than 1500 genes, many of which are expressed only in male germ cells. Expression of two-thirds of these depends both on a testis-specific version of the MMB (Myb-Muv B)/dREAM (Drosophila RBF, dE2F2, and dMyb-interacting proteins) complex termed the testis meiotic arrest complex (tMAC) and on testis-specific paralogs of TATA-binding protein-associated factors (tTAFs). Although this is one of the most dramatic changes in gene expression in Drosophila, it is not yet understood how the testis-specific transcripts are selectively activated during the 3-day spermatocyte period (Kim, 2017).

To identify the first transcripts up-regulated at onset of spermatocyte differentiation, germ cells were genetically manipulated to synchronously differentiate from spermatogonia to spermatocytes in vivo using bam^-/- testes, which contain large numbers of overproliferating spermatogonia. Brief restoration of Bam expression under heat shock control in hs-bam;bam^-/- flies induced synchronous differentiation of bam^-/- spermatogonia, resulting in completion of a final mitosis, premeiotic DNA synthesis, and onset of spermatocyte differentiation by 24 hours after Bam expression, eventually leading to production of functional sperm. Comparison by means of microarray of transcripts expressed before versus 24 hours after heat shock of hs-bam;bam^-/- testes identified 27 early transcripts that were significantly up-regulated more than twofold in testes from hs-bam;bam^-/- but not from bam^-/- flies subjected to the same heat shock regime. Among these was the early spermatocyte marker RNA binding protein 4 (Rbp4). At this early time point, the transcript for CG5204 - now named kumgang (kmg), from the Korean name of mythological guardians at the gate of Buddhist temples - had the greatest increase among all 754 Drosophila predicted transcription factors (Kim, 2017).

Kumgang (CG5204) encodes a 747-amino acid protein with six canonical C2H2-type zinc finger domains expressed in testes but not in ovary or carcass. Kmg protein was expressed independently from the tMAC component Always early (Aly) or the tTAF Spermatocyte Arrest (Sa), and both kmg mRNA and protein were up-regulated before Topi, another component of tMAC. Immunofluorescence staining of wild-type testes revealed Kmg protein expressed specifically in differentiating spermatocytes, where it was nuclear and enriched on the partially condensed bivalent chromosomes. Consistent with dramatic up-regulation of kmg mRNA after the switch from spermatogonia to spermatocyte, expression of Kmg was first detected with immunofluorescence staining after completion of premeiotic S-phase marked by down-regulation of Bam, coinciding with expression of Rbp4 protein (Kim, 2017).

Function of Kmg in spermatocytes was required for male germ cell differentiation. Reducing function of Kmg in spermatocytes-either by means of cell type-specific RNA interference (RNAi) knockdown (KD) or in flies trans-heterozygous for a CRISPR (clustered regularly interspaced short palindromic repeats)-induced kmg frameshift mutant and a chromosomal deficiency (kmg^Δ7/Df)-resulted in accumulation of mature primary spermatocytes arrested just before the G₂/M transition for meiosis I and lack of spermatid differentiation. A 4.3-kb genomic rescue transgene containing the 2.3-kb kmg open reading frame fully rescued the differentiation defects and sterility of kmg^Δ7/Df flies, confirming that the meiotic arrest phenotype was due to loss of function of Kmg. In both kmg KD and kmg^Δ7/Df, Kmg protein levels were less than 5% that of wild type. kmg^Δ7/Df mutant animals were adult-viable and female-fertile but male-sterile, which is consistent with the testis-specific expression (Kim, 2017).

Function of Kmg was required in germ cells for repression of more than 400 genes not normally expressed in wild-type spermatocytes. Although the differentiation defects caused by loss of function of kmg appeared, by means of phase contrast microscopy, to be similar to the meiotic arrest phenotype of testis-specific tMAC component mutants, analysis of gene expression in kmg KD testes showed that many Aly (tMAC)-dependent spermatid differentiation genes were expressed, although some at a lower level than that in wild type. Among the 652 genes with more than 99% lower expression in aly^-/- mutant as compared with wild-type testes, only four showed similar reduced expression in kmg KD as compared with that of sibling control (no Gal4 driver) testes. In contrast, transcripts from more than 500 genes were strongly up-regulated in kmg KD testes, with almost no detectable expression in testes from sibling control males. Hierarchical clustering identified 440 genes specifically up-regulated in kmg KD testes compared with testes from wild-type, bam^-/-, aly^-/-, or sa^-/- mutant flies. These 440 genes were significantly associated with Gene Ontology terms such as 'substrate specific channel activity' or 'detection of visible light' that appeared more applicable to non-germ cell types, such as neurons. Analysis of published transcript expression data for a variety of Drosophila tissues revealed that the 440 were normally not expressed or extremely low in wild-type adult testes, but many were expressed in specific differentiated somatic tissues such as eye, brain, or gut. Confirming misexpression of neuronal genes at the protein level, immunofluorescence staining revealed that the neuronal transcription factor Prospero (Pros), normally not detected in male germ cells, was expressed in clones of spermatocytes that are homozygous mutant for kmg induced by Flp-FRT-mediated mitotic recombination. The misexpression of Pros was cell-autonomous, occurring only in mutant germ cells. Mid-stage to mature spermatocytes homozygous mutant for kmg misexpressed Pros, but mutant early spermatocytes did not, indicating that the abnormal up-regulation of Pros occurred only after spermatocytes had reached a specific stage in their differentiation program (Kim, 2017).

A small-scale cell type-specific RNAi screen of chromatin regulators revealed that KD of dMi-2 in late TA cells and spermatocytes resulted in meiotic arrest, similar to loss of function of kmg. Immunofluorescence analysis of testes from a protein trap line in which an endogenous allele of dMi-2 was tagged by green fluorescent protein (GFP) revealed that dMi-2-GFP, like the untagged endogenous protein, was expressed and nuclear in progenitor cells and spermatocytes, as well as in somatic hub and cyst cells. dMi-2-GFP colocalized to chromatin with Kmg in spermatocytes, and the level of dMi-2 protein appeared lower and less concentrated on chromatin in nuclei of kmg^-/- spermatocytes than in neighboring kmg^+/+ or kmg^+/- spermatocytes, suggesting that Kmg may at least partially help recruit dMi-2 to chromatin in spermatocytes. Furthermore, in testis extracts Kmg coimmunoprecipitated with dMi-2 and vice versa, suggesting that Kmg and dMi-2 form a protein complex in spermatocytes. Comparison of microarray data revealed that most of the 440 transcripts up-regulated in testes upon loss of function of kmg were also abnormally up-regulated in dMi-2 KD testes, suggesting that Kmg and dMi-2 may function together to repress expression of the same set of normally somatic transcripts in spermatocytes (Kim, 2017).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that Kmg protein localized along the bodies of genes actively transcribed in the testis. ChIP-seq with antibody to Kmg identified 798 genomic regions strongly enriched by immunoprecipitation of Kmg from wild-type but not from kmg KD testes. Of the 798 robust Kmg ChIP-seq peaks, 698 overlapped with exonic regions of 680 different genes actively transcribed in testes. The enrichment was often strongest just downstream of the transcription start site (TSS), but with substantial enrichment along the gene body as well (Kim, 2017).

ChIP-seq with antibody to dMi-2 also showed enrichment along the gene bodies of the same 680 genes bound by Kmg, with a similar bias just downstream of the TSS. The dMi-2 ChIP signal along these genes was partially reduced in kmg KD testes, suggesting that Kmg may recruit dMi-2 to the bodies of genes actively transcribed in the testis (Kim, 2017).

RNA-seq analysis revealed that the 680 genes bound by Kmg were strongly expressed in testes and most strongly enriched in the GO term categories 'spermatogenesis' and 'male gamete generation'. One-third of the genes bound by Kmg were robustly activated as spermatogonia differentiate into spermatocytes and were much more highly expressed in the testes than in other tissues. The median levels of transcript expression of most of the 680 Kmg bound genes did not show appreciable change upon loss of Kmg (Kim, 2017).

Genes that are normally transcribed in somatic cells that became up-regulated upon loss of Kmg function in spermatocytes for the most part did not appear to be bound by Kmg. Only 3 of the 440 genes up-regulated in kmg KD overlapped with the 680 genes with robust Kmg peaks, suggesting that Kmg may prevent misexpression of normally somatic transcripts either indirectly or by acting at a distance (Kim, 2017).

Inspection of RNA-seq reads from kmg and dMi-2 KD testes mapped onto the genome showed that ~80% of the transcripts that were detected with microarray analysis as misexpressed in KD as compared with wild-type testes did not initiate from the promoters used in the somatic tissues in which the genes are normally expressed. Metagene analysis, as well as visualization of RNA expression centered on the TSSs annotated in the Ensembl database, showed that most of the 143 genes that are normally expressed in wild-type heads but not in wild-type testes were misexpressed in kmg or dMi-2 KD testes from a start site different from the annotated TSS used in heads. Transcript assembly from RNA-seq data by using Cufflinks for the 143 genes also showed that the transcripts that are misexpressed in kmg or dMi-2 KD testes most often initiate from different TSSs than the transcripts from the same gene assembled from wild-type heads (Kim, 2017).

Of the 440 genes scored via microarray as derepressed in kmg KD testes, 346 could be assigned with TSSs in kmg KD testes based on visual inspection of the RNA-seq data mapped onto the genome browser. Of these, only 67 produced transcripts in kmg KD testes that started within 100 base pairs (bp) of the TSS annotated in the Ensembl database, based on the tissue(s) in which the gene was normally expressed. In contrast, for the rest of the 346 genes, the transcripts expressed in kmg KD testes started from either a TSS upstream (131 of 346) or downstream (148 of 346) of the annotated TSSs. Of the 346 genes, 262 were misexpressed starting from nearly identical positions in dMi-2 KD as in kmg KD testes, suggesting that Kmg and dMi-2 function together to prevent misexpression from cryptic promoters (Kim, 2017).

Many of the ectopic promoters from which the misexpressed transcripts originated appeared to be bound by Aly, a component of tMAC, in kmg KD testes. ChIP for Aly was performed by using antibody to hemagglutinin (HA) on testis extracts from flies bearing an Aly-HA genomic transgene able to fully rescue the aly^-/- phenotype. Of 346 genes with new TSSs assigned via visual inspection, 181 had a region of significant enrichment for Aly as detected with ChIP, with its peak summit located within 100 bp of the cryptic promoter. Motif analysis by means of MEME revealed that these regions were enriched for the DNA sequence motif (AGYWGGC). This motif was not significantly enriched in the set of 165 cryptic promoters at which Aly was not detected in kmg KD testes. Enrichment of Aly at the cryptic promoters was much stronger in kmg KD as compared with wild-type testes, suggesting that in the absence of Kmg, Aly may bind to and activate misexpression from cryptic promoters (Kim, 2017).

Genetic tests revealed that the misexpression of somatic transcripts in kmg KD spermatocytes indeed required function of Aly. The neuronal transcription factor Pros, abnormally up-regulated in kmg KD or mutant spermatocytes, was no longer misexpressed if the kmg KD spermatocytes were also mutant for aly, even though germ cells in kmg KD;aly^-/- testes appear to reach the differentiation stage at which Pros turned on in the kmg KD germ cells. Assessment by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that misexpression of five out of five transcripts in kmg KD testes also required function of Aly. Global transcriptome analysis via microarray of kmg KD versus kmg KD;aly^-/- testes showed that the majority of the 440 genes that were derepressed because of loss of function of kmg in spermatocytes were no longer abnormally up-regulated in kmg KD;aly^-/- testes. Even genes without noticeable binding of Aly at their cryptic promoters were suppressed in kmg KD;aly^-/-, suggesting that Aly may regulate this group of genes indirectly (Kim, 2017).

Together, the ChIP and RNA-seq data show that Kmg and dMi-2 bind actively transcribed genes but are required to block expression of aberrant transcripts from other genes that are normally silent in testes. The mammalian ortholog of dMi-2, CHD4 (Mi-2β), has been shown to bind active genes in mouse embryonic stem cells or T lymphocyte precursors but also plays a role in ensuring lineage-specific gene expression in other contexts. It cannot be ruled out that Kmg and dMi-2 might also act directly at the cryptic promoter sites but that the ChIP conditions did not capture their transient or dynamic binding because several chromatin remodelers or transcription factors, such as the thyroid hormone receptor, have been difficult to detect with ChIP. Kmg and dMi-2 may repress misexpression from cryptic promoters indirectly by activating as-yet-unidentified repressor proteins. However, it is also possible that Kmg and dMi-2 act at a distance by modulating chromatin structure or confining transcriptional initiation or elongation licensing machinery to normally active genes (Kim, 2017).

Changes in the genomic localization of Aly protein in wild-type versus kmg KD testes raised the possibility that Kmg may in part prevent misexpression from cryptic promoters by concentrating Aly at active genes. Of the 1903 Aly peaks identified with ChIP from wild-type testes, the 248 Aly peaks that overlapped with strong Kmg peaks showed via ChIP an overall reduction in enrichment of Aly from kmg KD testes as compared with wild type. In contrast, the Aly peaks at cryptic promoters were more robust in kmg KD testes than in wild type. In general, over the genome 4129 new Aly peaks were identified by means of ChIP from kmg KD testes that were absent or did not pass the statistical cutoff in wild-type testes. More than 30% of the genomic regions with new Aly peaks in kmg KD showed elevated levels of RNA expression starting at or near the Aly peak in kmg KD but not in wild-type testes, suggesting that misexpression of transcripts from normally silent promoters in kmg KD testes is more widespread than initially assessed with microarray. Together, these findings raise the possibility that Kmg may prevent misexpression of aberrant transcript by concentrating Aly to active target genes in wild-type testes, preventing binding and action of Aly at cryptic promoter sites (Kim, 2017).

The results suggest that selective gene activation is not always mediated by a precise transcriptional activator but can instead be directed by combination of a promiscuous activator and a gene-selective licensing mechanism. Cryptic promoters may become accessible as chromatin organization is reshaped to allow expression of terminal differentiation transcripts that were tightly repressed in the progenitor state. It is posited that this chromatin organization makes a number of sites that are accessible for transcription dependent on the testis-specific tMAC complex component Aly. In this context, activity of Kmg and dMi-2 is required to prevent productive transcript formation from unwanted initiation sites, potentially by confining Aly to genes actively transcribed in the testis and limiting the amount of Aly protein acting at cryptic promoters (Kim, 2017).

The initiation of transcripts from cryptic promoters is reminiscent of loss of function of Ikaros, a critical regulator of T and B cell differentiation and a tumor suppressor in the lymphocyte lineage. Like Kmg, Ikaros is a multiple-zinc finger protein associated with Mi-2β, which binds to active genes in T and B cell precursors. In T cell lineage acute lymphoblastic leukemia (T-ALL) associated with loss of function of Ikaros, cryptic intragenic promoters were activated, leading to expression of ligand-independent Notch1 protein, contributing to leukemogenesis. Thus, in addition to being detrimental for proper differentiation, firing of abnormal transcripts from normally cryptic promoters because of defects in chromatin regulators may contribute to tumorigenesis through generation of oncogenic proteins (Kim, 2017).

Reactive oxygen species (ROS) are byproducts generated during normal cellular metabolism, and redox states have been shown to influence stem cell self-renewal and lineage commitment across phyla. However, the downstream effectors of ROS signaling that control stem cell behavior remain largely unexplored. This study used the Drosophila testis as an in vivo model to identify ROS-induced effectors that are involved in the differentiation process of germline stem cells (GSCs). In the Affymetrix microarray analysis, 152 genes were either upregulated or downregulated during GSC differentiation induced by elevated levels of ROS, and a follow-up validation of the gene expression by qRT-PCR showed a Spearman's rho of 0.9173 (P<0.0001). Notably, 47 (31%) of the identified genes had no predicted molecular function or recognizable protein domain. These suggest the robustness of this microarray analysis, which identified many uncharacterized genes, possibly with an essential role in ROS-induced GSC differentiation. maf-S was shown to be transcriptionally downregulated by oxidative stress, and maf-S knockdown promotes GSC differentiation, but Maf-S overexpression conversely results in an over-growth of GSC-like cells by promoting the mitotic activity of germ cell lineage. Together with the facts that Maf-S regulates ROS levels and genetically interacts with Keap1/Nrf2 in GSC maintenance, this study suggests that Maf-S plays an important role in the Drosophila testis GSC maintenance by participating in the regulation of redox homeostasis (Tan, 2017).

Genotoxic stress such as irradiation causes a temporary halt in tissue regeneration. The ability to regain regeneration depends on the type of cells that survived the assault. Previous studies showed that this propensity is usually held by the tissue-specific stem cells. However, stem cells cannot maintain their unique properties without the support of their surrounding niche cells. This study shows that exposure of Drosophila melanogaster to extremely high levels of irradiation temporarily arrests spermatogenesis and kills half of the stem cells. In marked contrast, the hub cells that constitute a major component of the niche remain completely intact. It was further shown that this atypical resistance to cell death relies on the expression of certain antiapoptotic microRNAs (miRNAs) that are selectively expressed in the hub and keep the cells inert to apoptotic stress signals. It is proposed that at the tissue level, protection of a specific group of niche cells from apoptosis underlies ongoing stem cell turnover and tissue regeneration (Volin, 2018).

Cancer stem cells, in contrast to their more differentiated daughter cells, can endure genotoxic insults, escape apoptosis, and cause tumor recurrence. Understanding how normal adult stem cells survive and go to quiescence may help identify druggable pathways that cancer stem cells have co-opted. This study utilized a genetically tractable model for stem cell survival in the Drosophila gonad to screen drug candidates and probe chemical-genetic interactions. This study employs three levels of small molecule screening: a medium-throughput primary screen in male germline stem cells (GSCs), a secondary screen with irradiation and protein-constrained food in female GSCs, and a tertiary screen in breast cancer organoids in vitro. This study uncover a series of small molecule drug candidates that may sensitize cancer stem cells to apoptosis. This study assessed these small molecules for chemical-genetic interactions in the germline and identified the NF-kappaB pathway as an essential and druggable pathway in GSC quiescence and viability. This study demonstrates the power of the Drosophila stem cell niche as a model system for targeted drug discovery (Ishibashi, 2021).

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. This study identified fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. Testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification (Iki, 2020).

Gene silencing mediated by non-coding small RNAs (sRNAs) underlies diverse biological processes, including development, homeostasis, immunity, and reproduction in eukaryotes. Lines of evidence indicate that the expression and steady-state accumulation of regulatory sRNAs themselves are controlled by multiple, intertwined mechanisms, the misregulation of which has been implicated in disease. However, understanding the regulation of sRNA expression in vivo is still incomplete (Iki, 2020).

The sRNAs cooperate with Argonaute (Ago) family proteins, forming the core effector, namely, RNA-induced silencing complex (RISC). Many sRNAs are generated through the processing of double-stranded RNAs (dsRNAs) by RNase III enzymes. Most microRNAs (miRNAs) are derived from imperfectly base-paired hairpin structures within non-coding transcripts or intronic fragments, whereas short interfering RNAs (siRNAs) originate from highly base-paired dsRNA of both endogenous and exogenous origins. Cleavages by RNase III excise ~20 to 24-nucleotide (nt) mi/siRNAs as duplex intermediates, which upon loading onto Ago proteins, become unwound during RISC formation. One strand is selectively stabilized as a 'guide strand' that recognizes the target RNA displaying sRNA-complementary sites, enabling gene silencing. The complementary so called 'passenger strand,' by contrast, is eliminated. Depending on various cell/tissue/organ-specific features, including but not restricted to sRNA alternative processing, 3'-end tailing, or even editing, a guide strand can become a passenger strand and vice versa. Hence, RISC formation represents one one of the key steps determining the steady-state accumulation of specific sRNA cohorts in vivo (Iki, 2020).

Previous studies in both animals and plants demonstrated the fundamental requirement of the Hsp70/90 chaperone machinery for RISC formation. One of the core factors, Hsp90, is of particular importance by retaining Ago in an 'apo' state, an open conformation competent for sRNA duplex incorporation. By an open-close cycle driven by ATP binding and its hydrolysis, Hsp90 chaperone homodimers transiently interact with selected client proteins as well as distinct co-chaperones at particular steps. Hsp90 then assists multiple reactions, including the binding of small ligands to client proteins. As one of Hsp90 co-chaperones, Cyclophilin 40 (CYP40)/SQUINT promotes miRNA activities in plants by facilitating the binding of duplexes (ligands) to Ago1 (client). In mammalian cells, other Hsp90 co-chaperones, namely, FK506-binding protein (Fkbp)4/Fkbp52 and the homolog Fkbp5/Fkbp51, interact with Ago2 to promote RISC formation. However, a broader range of ligand-client binding reactions can be controlled by these Hsp90 co-chaperones. Indeed, both Cyp40 and Fkbp52 were first isolated as cofactors of nuclear receptors (clients) binding to steroid hormones (ligands) in animals. These interactions were functionally characterized in various studies involving cultured mammalian cells, yeast systems, or purified proteins. Consistently, Fkbp52 knockout mice exhibit reproductive defects accompanied with attenuated steroid sensitivity. In contrast, Fkbp52 Fkbp51 double-knockout individuals die in the early embryonic stage, implying other important, yet uncharacterized, functions for both co-chaperones, possibly including a role in RISC formation. However, the biological significance of Fkbp52- and Fkbp51-regulated RISC formation has yet to be addressed in vivo, and likewise, Cyp40 knockout animals have been unavailable except in the case of a protist model. Consequently, physiological functions for animal Cyp40 remain largely elusive (Iki, 2020).

Drosophila melanogaster provides an excellent model to study the commonalities and specificities of the miRNA versus siRNA pathways. Among the five Ago protein members encoded in the fly genome, Ago1 and Ago2 incorporate RNase III Dicer 1 (Dcr1)-dependent miRNA and Dcr2-dependent siRNA duplexes. The other three Ago proteins are dedicated to the loading of Piwi-interacting RNA accumulating prominently in the gonads in a Dcr-independent manner. Both structural and nucleotide sequence features of mi/siRNA duplexes underpin their sorting into Ago1 or Ago2, as well as the subsequent guide-strand selection process. In this respect, fly Ago1 hosts a wide range of miRNAs and is essential for development from early embryogenesis. In contrast, fly Ago2 is primarily required for antiviral defense by interacting with virus-derived siRNAs. Independent of its defensive roles, Ago2 is also loaded with endogenous siRNA (endo-siRNA) in testes to support the male reproductive functions. Apart from viral and endo-siRNAs, a smaller and selective fraction of miRNAs is preferentially sorted into Ago2 rather than Ago1, although the biological roles of these rarer Ago2-bound miRNAs has remained largely unexplored (Iki, 2020).

Whether, in addition to their key role in activating the apo-Ago conformation, (co-)chaperones exhibit RISC regulatory activities influencing sRNA steady-state accumulation remains an open question, as does the potential tissue- or cell-type specificity of these putative regulatory functions. To explore these issues and, more generally, better understand the physiological roles of Hsp90 co-chaperones in animals, the loss-of-function alleles of cyp40, fkbp4, and fkbp5 orthologs were generated and characterized in D. melanogaster. Although the fkbp mutant died at the pupal stage, the cyp40 mutant was eclosed but the male exhibited infertility, a phenotype resembling that of the ago2 mutant. These detailed analyses of Ago-bound sRNAs in testes show that Cyp40 is selectively required for the accumulation of testis-unique and functional miRNAs sorted to Ago2. The significance of these results is discussed in a broader context of how regulation on specific sRNA repertoires might be enabled by tissue-specialized chaperone expression (Iki, 2020).

This study uncovered an essential function of Cyp40 as an Hsp90 co-chaperone during late spermatogenesis in Drosophila. The in vitro physical interaction analyses and in vivo Ago-bound sRNA profiling revealed a molecular activity of Cyp40 in regulating the steady-state accumulation of miRNAs uniquely forming RISC with Ago2 in testes. Although the Drosophila miRNA-sorting mechanism is now well established, the biological basis underlying the binding of a cohort of miRNAs to Ago2 had remained elusive. This study has uncovered one such mechanism and biological circumstance by showing how, under the control of germline Cyp40, Ago2 efficiently binds testis-unique miRNAs. Because the genetic ablation of one of these miRNAs suffice to noticeably impair proper spermatid differentiation, these findings also support the notion that the miRNA-Ago2 sorting pathway, which is conserved in Drosophila, is physiologically relevant (Iki, 2020).

Although miRNA hairpins are usually considered to spawn a single dominant guide strand, previous studies reported guide-strand alteration, or arm switching, in a tissue- and/or developmental-stage-specific manner. This study has identified several such miRNAs that are subjected not only to altered guide-strand selection but also to unconventional Ago sorting in a testis-unique manner. It was further demonstrated the functional relevance of these alternative events with the example of miR-316. What mechanisms might underlie the strand/Ago-switching phenomenon? The central unpaired nucleotides, terminal instabilities of sRNA duplexes, and 5' nucleotide identities largely govern, together, Ago sorting and strand selection in Drosophila. In both plants and metazoans, such duplex-intrinsic features are, however, modifiable through alternative precursor processing by RNase III enzymes. As a result, excised duplex isoforms acquire novel fates through RISC formation. In the case of miR-316, the majority of strands share an identical 5' end, reflecting a fixed, as opposed to flexible, processing mechanism. Thus, the switching strand-selection behavior of miR-316 is most likely attributable to other mechanisms. They could include RNA editing, as observed in mammals; although, testing this possibility in flies will require further work. Notably, loss of cyp40 function perturbed Ago2 binding to miR-316-3p, and yet, neither abrogated the 3'-over-5' strand preference nor affected Ago1 sorting. Hence, Cyp40 might in part influence the 5'/3'-strand use bias, but it is unlikely to regulate strand switching per se (Iki, 2020).

Besides these considerations of the strand selection of some miRNA species, Cyp40 had a profound, global effect on discriminating miRNAs from endo-siRNAs bound to Ago2. In flies, miRNA and siRNA duplexes are delivered to Ago2 by loading complexes constituted of Dcr-2 and co-partners R2D2 or Loqs-PD. By asymmetry sensing, the complexes preferentially bind near-perfectly base-paired siRNA duplexes to enable their efficient loading, whereas they simultaneously disfavor duplexes containing central instability, a feature of many miRNAs. The process is thought, among other possibilities, to avoid the saturation of Ago2 by miRNAs during virus infections, which would presumably compromise siRNA-mediated antiviral defenses. This circumstance added to the fact that many arthropod viruses encode silencing suppressor proteins probably explain why Ago2 and Dcr2, unlike Ago1 and Dcr1, are among the fastest evolving 3% of all Drosophila genes, owing to positive selection as a consequence of the never ending host-pathogen arms race. Nonetheless, these results are in line with the observation that the Drosophila argo2 gene has also evolved under the adaptive basis of testis specialization, most likely independently of antiviral roles. Indeed, as much as Ago2-bound hp-siRNAs have co-evolved with target genes and assist male fertility, the data suggest that miRNAs, such as miR-316, can also gain functional roles through unconventional Ago2 sorting in testes. However, as typical miRNAs, Cyp40-dependent duplexes displayed central bulges or mismatches, of which a likely consequence is their predicted low loading efficacy into Ago2. This might have constituted one of the driving forces for the deployment of a germline Cyp40, which, together with Hsp90, likely improves the affinity of Ago2 for the kinetically less favorable miRNA members (Iki, 2020).

Together with the TPR domain tethering Cyp40 to Hsp90 during RISC formation, the PPIase domain is required to form functional chaperone complexes in both flies and plants. The PPIase domain of human Cyp40/PPID maintains the activity for the cis-trans isomerization of peptidyl prolines. Further biochemical and structural analyses could help decipher how this, and perhaps other activities of the poorly characterized PPIase domain, enable fly and plant Cyp40 to regulate sRNA during RISC formation. In addition, Fkbp, among the Hsp90 co-chaperones regulating ligand-client binding, displays a similar domain architecture to Cyp40, but endows distinct physiological functions. The generated fkbp59-mutant-based study should help illuminate the molecular basis for such a difference (Iki, 2020).

Why is Cyp40 effective with Ago2 rather than Ago1? In testes, Ago2 appears to be relatively limited compared to Ago1, whereas its main endogenous cargoes, hairpin-siRNA duplexes, are actively produced. This likely creates a competition between miRNA and siRNA duplexes for a limited pool of Ago2. In the duplex-competing condition, Cyp40 activity could ensure Ago2 loading of suboptimal miRNA duplexes, which would otherwise be largely excluded from the effector phase of the testicular silencing pathway. However, Ago1 is inert for most endo-siRNAs and appears to be relatively abundant, compared to Ago2, in testes. This might allow Ago1 to naturally evade the testicular duplex-competing condition, explaining why Cyp40 interacts with Ago1 in vitro and yet is dispensable for steady-state accumulation of most Ago1-bound miRNAs in testes. Thus, the relative abundance of duplexes compared to that of the sRNA-free Ago proteins could be key for influencing the selectivity and effectiveness of Cyp40. The same rationale could also apply to other Hsp90 co-chaperones involved in RISC formation and sRNA regulation in other biological contexts and/or tissues (Iki, 2020).

The cyp40 mutant male showed a completely sterile phenotype, which appears to be exerted by the combined deficiencies of Ago2-sorted multiple miRNAs, including, but not restricted to, miR-316-3p. Similar to cyp40, argo2 mutant males are sterile in Drosophila simulans but are only semi-fertile in D. melanogaster, raising the question of how the loss of an Ago2 modulator, Cyp40, might cause a more severe phenotype than the loss of Ago2 itself. Perhaps the mis-accumulation of some sRNAs and the ensuring disparities among RISCs in the cyp40 mutant have deeper detrimental consequences than those caused by the mere depletion of the effector Ago2. For instance, it was shown that the reduction of selected miRNAs over-activates, in turn, other species, possibly engaging in a whole new boast of not primarily intended regulations. However, the possibility is not excluded that Cyp40 plays multiple roles in testes, in addition to the control of sRNA bound to Ago2. Also noteworthy as a caveat-inducing limitation of the approach taken in this study, Ago-bound sRNAs were isolated from whole testes, including somatic cells where cyp40 is dispensable. Refining this study using sRNA profiles from the purified germline cells, especially from the spermatocyte and spermatid stages, would likely confer more sensitivity to sRNA analyses and help more comprehensively decipher the sRNA-related molecular aberrations caused by loss of cyp40 function in the germline (Iki, 2020).

Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation, nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette, a temporary structure that assists in sperm elongation, plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. This study describes the role of an uncharacterized protein in Drosophila, Salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has a dynamic localization during spermatid differentiation, being progressively relocated from the sperm nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto null male flies are sterile and exhibit complete spermatid individualization defects. salto deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. This work sheds light on a novel component involved in cytoskeleton based cell morphological changes during spermiogenesis (Augiere, 2019).

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. This study elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Key roles were identified of nucleoporins and V-ATPase proton pumps, and their importance was demonstrated in controlling germline development from the support side. To make the dataset publicly available, an interactive analysis tool was generated, that uncovered conserved core genes of adult stem cells across species boundaries. The functional relevance of these genes was tested in the Drosophila testis and intestine, and a high frequency of stem cell defects was found. In summary, this dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types (Tamirisa, 2018).

Using cell-type-specific transcriptome profiling and in vivo TF binding site mapping together with an interactive data analysis tool, this study comprehensively identified genes involved in controlling proliferation and differentiation within a stem cell support system. Importantly, many candidates that were functionally tested not only were required within the soma, but also had non cell-autonomous functions in the adjacent (germline) stem cell lineage (Tamirisa, 2018).

An interconnected network of TFs was identified that plays an important role in the maintenance and differentiation of both germline and somatic cell populations, signal processing V-ATPase proton pumps, and nuclear-transport-engaged Nups as regulators in the Drosophila male stem cell system. V-ATPases have been implicated in the regulation of various cellular processes in not only invertebrates but also vertebrates. For example, the V-ATPase subunit V1e1 was previously shown to be essential for the maintenance of NBs in the developing mouse cortex, as loss of this subunit caused a reduction of endogenous Notch signaling and a depletion of NBs by promoting their differentiation into neurons. Furthermore, two independent studies revealed that V-ATPase subunits and their isoforms are required for proper spermatogenesis in mice, in particular for acrosome acidification and sperm maturation. Thus, it is tempting to speculate that these proton pumps also have important functions in the stem cell pool of the mammalian testis and very likely many other stem cell systems, and some evidence is provided for their crucial role also in ISCs (Tamirisa, 2018).

This work also uncovered nuclear transport associated proteins, the Nups, as important control hubs in the somatic lineage of the Drosophila testis. This is of particular interest, since cell-type-specific functions of Nups have been identified only recently and may represent a critical feature of different stem cell systems. Examples include Nup153, one of the Nups expressed in all four stem cell systems, which interacts with Sox2 neural progenitors and controls their maintenance as well as neuronal differentiation; Nup358, which plays a role at kinetochores, and Nup98, which regulates the anaphase promoting complex (APC) and mitotic microtubule dynamics to promote spindle assembly. Interestingly, it has been shown just recently that Nups play a critical role in regulating cell fate during early Drosophila embryogenesis, thereby contributing to the commitment of pluripotent somatic nuclei into distinct lineages. Current results suggest that they may play a similar role in controlling the transition of continuously active adult stem cells toward differentiation. The next challenge will be to unravel how variations in the composition of an essential and basic protein complex like the NPC causes differential responses of cells, in particular in stem cells and their progenies (Tamirisa, 2018).

The datasets in conjunction with the versatile and easy-to-use analysis tool allowed identification of a substantial number of stem cell regulators for detailed mechanistic characterization. Importantly, this analyses have shed first light on processes and genes shared between diverse invertebrate and vertebrate stem cell systems and uncovered functionally relevant differences. Owing to its flexibility and the option to include datasets from any species, the online tool represents a valuable resource for the entire stem cell community. It not only provides an open platform for data analysis but also leverages the power of comparative analysis to enable researchers mining genomic datasets from diverse origins in a meaningful and intuitive fashion (Tamirisa, 2018).

Adult stem cells are often found in specialized niches, where the constituent cells direct self-renewal of their stem cell pool. The niche is therefore crucial for both normal homeostasis and tissue regeneration. In many mammalian tissues, niche cells have classically been difficult to identify, which has hampered any understanding of how tissues first construct niches during development. Fortunately, the Drosophila germline stem cell (GSC) niche is well defined, allowing for unambiguous identification of both niche cells and resident stem cells. The testis niche: this study followed pro-niche cells as they assemble and assume their final form. After ex vivo culture the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent GSCs, and to direct GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. Dynamic processes were identified that constitute an assembly phase and a compaction phase during morphogenesis. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction, an assembly phase involves the movement of pro-niche cells along the outer periphery of the gonad, using the extracellular matrix (ECM) to assemble at the anterior of the gonad. Finally, live-imaging in integrin mutants allows definition of the role of pro-niche cell-ECM interaction with regard to the new assembly and compaction dynamics revealed in this study (Anllo, 2018).

This work is the first to characterize the dynamics of testis niche morphogenesis. Through live-imaging both in vivo and ex vivo, it was possible to follow pro-niche cells as they assemble and assume the final compact form of this niche. After ex vivo culture, the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent germline cells, and to direct the now GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction occurs, an assembly phase involves the movement of PS11 pro-niche cells along the outer periphery of the gonad, using the ECM to assemble at the anterior of the gonad. There they join PS10 pro-niche cells, forming an anterior and internally-facing cap. Finally, live-imaging in integrin mutants allows definition of the role of pro-niche cell-ECM interaction more precisely with regard to the new assembly and compaction dynamics revealed in this study (Anllo, 2018).

Previously, analysis of fixed samples nicely inferred a change from an initial cap of pro-hub cells to a compact niche. The current ex vivo imaging now visualizes compaction directly, and by comparing cell numbers at assembly and after compaction, shows that the initial assemblage of the elongate cap of cells indeed constitutes the pro-hub. Scoring the same gonad before and after compaction showed little, if any, shuffling in the number of pro-niche cells. This finding is consistent with the fact that specification of niche cells occurs much earlier in development, before the stages imaged in this study. Lastly, the current work suggests two cell biological phenomena that correlate with the process of compaction (Anllo, 2018).

Firstly, while compaction is underway, there is a burst of GSC divisions orthogonal to the hub. Interestingly, at late anaphase, the niche cells adjacent to a dividing GSC occasionally appeared to become compressed against their niche cell neighbors, subsequently occupying less space. It is known that activation of the STAT pathway is necessary to induce germline divisions in the (male) gonad. Since the ligand for pathway activation is secreted by hub cells, this suggests a possible scenario where niche cells orient GSC divisions, which subsequently generate some of the force necessary for niche compaction, pushing hub cells closer together. In principle, this scenario can now be tested by attempting to block GSC divisions during ex vivo culture (Anllo, 2018).

Secondly, hub compaction occurs concurrently with cell neighbor exchanges. These exchanges are reminiscent of convergence and extension, where directed junctional re-organization changes the overall shape of the sheet of cells. For example, at assembly, an individual cell within the elongate cap will shrink one of its boundaries, allowing cells that were previously on either side of this cell to form new contacts adhering to one another. Such events may impart an increasingly more compact overall shape to the incipient niche. In addition to lateral neighbor exchange, ingression was observed of some pro-niche cells during compaction. This results in stratification of the niche, where a few internal pro-hub cells now lie more internal to the superficial cap of peripheral hub cells. These lateral and stratifying exchanges are complex, but their comprehensive cataloging, now enabled by ex vivo imaging, may well reveal patterns of cell movements. In turn, those patterns will likely suggest specific circuits to test as regulators of compaction (Anllo, 2018).

The neighbor exchange behavior associated with compaction is reminiscent of cell sorting, where 'like' cells will maximize contacts among themselves, while minimizing those with different neighbor types. Indeed, imaging describes a significant smoothening of the boundary between the pro-niche and the tier of stem cells, an outcome expected from sorting phenomena. Increased accumulation of adhesion proteins such as ECadherin, NCadherin and Fasciclin III, has been noted at the Assembly stage, although manipulation of these did not appear to grossly affect compaction. While unidentified factors or significant redundancy among these might drive sorting, the live-imaging parameters established here now enable analysis of the dynamics of the process. Future work might reveal changes in, for example, timing of compaction and smoothening, rather than relying on endpoint analysis alone (Anllo, 2018).

The positioning of the niche at the gonad anterior could, in principle, be driven by a gonad-intrinsic mechanism. For example, SGPs across the gonad have different antero-posterior identities, as they originate from three separate parasegments (ps10, ps11, and ps12). Thus, they accumulate Hox proteins to differing levels, and AbdB mutant embryos can exhibit defects in hub position. However, it remains possible that these defects could be caused by broader patterning changes in tissues outside the gonad, rather than reflecting an intrinsic requirement for AbdB. Still, the fact that pro-hub cells are enriched for adhesion proteins from the assembly stage onward is consistent with the idea that sorting driven by homophilic adhesions could be a main driver for niche formation. Nevertheless, two novel observations from live-imaging strongly suggest the existence of an extra-gonadal influence (Anllo, 2018).

First, while the niche assembles at the anterior, it was found that it exhibits a consistent tilt towards the interior of the embryo. This biased placement of the hub towards internal organs suggests that an extra-gonadal cue is influencing niche position. Live-imaging did reveal tissues located near cells of the assembling pro-niche: a branch of the trachea, and an alary muscle. The ablations that were conducted rule out any major impact from these two tissues on niche formation. However, other tissues, such as the nearby gut and its surrounding musculature, remain to be tested (Anllo, 2018).

Second, the individual movements of PS11 cells to join the PS10 cohort suggests that homotypic adhesions and sorting alone cannot explain the position of assembly. During the stages leading up to those shown in this study, SGPs exhibit robust cytoplasmic extensions that probe the internal milieu of the newly formed gonad, tightly embracing each other, and germline neighbors. Indeed, imaging carried out in this study revealed PS11 cellular extensions around germ cells at the onset of assembly. If sorting were the main driver for assembly, these extensions should be sufficient to cause PS11 cells to move through the internal gonad milieu, seek out and coalesce with anteriorly located PS10 SGPs. Instead, PS11 cells move out to the gonad periphery, and retract their trailing extensions. They then move along the periphery towards the anterior. In addition, if PS 11 cells were sorting together one might have expected these pro-niche cells to move as one tight collective, reminiscent of how the somatic border cell cluster migrates through the internal milieu of the Drosophila egg chamber. In contrast, the PS11 cells often move individually or as a string of cells along the periphery. Taken together, PS 11 cell behavior is much more in line with individual cell migration directed by a cue (Anllo, 2018).

Previous work described an ECM visible at the gonad periphery at the end of embryogenesis. Pigment cells ensheath the gonad, and are the likely source of this ECM. Indeed, live-imaging revealed that this ECM is present while PS11 cells are moving anteriorly. Thus, it is suspected that the matrix provides traction for movement in response to the posited external guidance cue (Anllo, 2018).

In fact, previous work has suggested a role for ECM interaction, since in the absence of Integrin function, the newly formed hub was located inside the gonadal sphere (Tanentzapf, 2007). Since that work had to rely on endpoint analysis, it was reasonable to assume that the hub had fully formed at the gonad anterior and then was internalized whole due to lack of Integrin-based adhesion. Instead, the current live-imaging of Integrin mutants now reveals that Integrin-ECM interactions are necessary well before compaction, as pro-niche cells can leave the periphery of the gonad before the niche compacts. This is the case for PS10 cells, which can be observed internalizing from the anterior of the gonad before compaction, and for PS11 cells, either after they arrive at the anterior or directly internalizing from their more posterior, PS11 location. Hub compaction occurs in Integrin mutants (Tanentzapf, 2007), but the current imaging reveals this to occur mostly after the niche internalizes in the mutants. The observation that PS11 cells can directly join an internalized cluster suggests that, left to their own, pro-hub cells might sort to inappropriate locations due to preferential adhesion. This implies that Integrin-ECM interactions at the periphery antagonize the sorting tendency of pro-niche cells. If Integrin-ECM interactions during migration are compromised, the resulting testis would exhibit aberrant polarity, as the niche would not be localized at the closed end of the tubule. Although some of the components making up the ECM surrounding the gonad have been characterized, which of these engage Integrins on the pro-niche cells is not clear (Anllo, 2018).

The nature of the directional cue read by PS11 cells is not known. Imaging shows that some but not all PS11 cells successfully arrive at the anterior in Integrin mutants. This suggests that pro-niche cells have the capability to respond to the cue, at least partially, even if they cannot interact with peripheral ECM. Normally, however, since all the PS11 cells that were tracked in wild type gonads migrated to the anterior along the ECM at the periphery of the gonad, these Integrin-ECM interactions serve to ensure reproducible positioning of the niche (Anllo, 2018).

The combination of in vivo and ex vivo imaging presented in this study overcomes previous challenges to understanding how a stem cell niche is formed. Visualizing the dynamics of niche formation has proven powerful in suggesting unanticipated mechanisms for niche morphogenesis in this work. This imaging should now allow for in-depth study of both the process of assembly and compaction into a fully functioning stem cell niche (Anllo, 2018).

Germ cell maturation is essential for spermatogenesis and testis homeostasis. ATP synthase serves significant roles in energy storage in germ cell survival and is catalyzed by alterations in the mitochondrial membrane proton concentration. The intrinsic cellular mechanisms governing stem cell maturation remain largely unknown. In the present study, in vivo RNA interference (RNAi) screening of major ATP synthase subunits was performed, and the function of ATP synthase for male fertility and spermatogenesis in Drosophila was explored. A Upstream Activation Sequence/Gal4 transcription factor system was used to knock down gene expression in specific cell types, and immunofluorescence staining was conducted to assess the roles of ATP synthase subunits in Drosophila testes. It was identified that knockdown of ATP synthase resulted in male infertility and abnormal spermatogenesis in Drosophila testes. In addition, knockdown of the ATP synthase beta subunit in germ cells resulted in defects in male infertility and germ cell maturation, while the hub and cyst cell populations were maintained. Other major ATP synthase subunits were also examined and similar phenotypes in Drosophila testes were identified. Taken together, the data from the present study revealed that ATP synthase serves important roles for male fertility during spermatogenesis by regulating germ cell maturation in Drosophila testes (Yu, 2019).

Stem cell niche is regulated by intrinsic and extrinsic factors. In the Drosophila testis, cyst stem cells (CySCs) support the differentiation of germline stem cells (GSCs). However, the underlying mechanisms remain unclear. This study found that somatic CG6015 is required for CySC maintenance and GSC differentiation in a Drosophila model. Knockdown of CG6015 in CySCs caused aberrant activation of dpERK in undifferentiated germ cells in the Drosophila testis, and disruption of key downstream targets of EGFR signaling (Dsor1 and rl) in CySCs results in a phenotype resembling that of CG6015 knockdown. CG6015, Dsor1, and rl are essential for the survival of Drosophila cell line Schneider 2 (S2) cells. The data showed that somatic CG6015 regulates CySC maintenance and GSC differentiation via EGFR signaling, and inhibits aberrant activation of germline dpERK signals. These findings indicate regulatory mechanisms of stem cell niche homeostasis in the Drosophila testis (Zheng, 2021).

Tissue-specific stem cells maintain tissue homeostasis by providing a continuous supply of differentiated cells throughout the life of organisms. Differentiated/differentiating cells can revert back to a stem cell identity via dedifferentiation to help maintain the stem cell pool beyond the lifetime of individual stem cells. Although dedifferentiation is important for maintaining the stem cell population, it is speculated that it underlies tumorigenesis. Therefore, this process must be tightly controlled. This study shows that a translational regulator, me31B, plays a critical role in preventing excess dedifferentiation in the Drosophila male germline: in the absence of me31B, spermatogonia dedifferentiate into germline stem cells (GSCs) at a dramatically elevated frequency. These results show that the excess dedifferentiation is likely due to misregulation of nos, a key regulator of germ cell identity and GSC maintenance. Taken together, the data reveal negative regulation of dedifferentiation to balance stem cell maintenance with differentiation (Jensen, 2021).

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1 (CG4078), was identified as a novel regulator of male germline stem cells (GSCs). Genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, these findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1 (Yang, 2021).

In Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. This study found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, it was observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, it is proposed that male-biased protein synthesis may be responsible for the sex differences observed in the early germline (Masukawa, 2021).

The Drosophila GAGA-factor encoded by the Trithorax-like (Trl) gene is DNA-binding protein with unusually wide range of applications in diverse cell contexts. In Drosophila spermatogenesis, reduced GAGA expression caused by Trl mutations induces mass autophagy leading to germ cell death. This work investigated the contribution of mitochondrial abnormalities to autophagic germ cell death in Trl gene mutants. Using a cytological approach, in combination with an analysis of high-throughput RNA sequencing (RNA-seq) data, it was demonstrated that the GAGA deficiency led to considerable defects in mitochondrial ultrastructure, by causing misregulation of GAGA target genes encoding essential components of mitochondrial molecular machinery. Mitochondrial anomalies induced excessive production of reactive oxygen species and their release into the cytoplasm, thereby provoking oxidative stress. Changes in transcription levels of some GAGA-independent genes in the Trl mutants indicated that testis cells experience ATP deficiency and metabolic aberrations, that may trigger extensive autophagy progressing to cell death (Dorogova, 2023).

This study provides a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. Assignment of key germline and somatic cell types was assigned using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, datasets compatible with commonly used software such as Seurat and Monocle is provided. The foundation provided in this study will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo (Raz, 2023).

A unifying feature of polycystin-2 channels is their localization to both primary and motile cilia/flagella. In Drosophila melanogaster, the fly polycystin-2 homologue, Amo, is an ER protein early in sperm development but the protein must ultimately cluster at the flagellar tip in mature sperm to be fully functional. Male flies lacking appropriate Amo localization are sterile due to abnormal sperm motility and failure of sperm storage. A forward genetic screen was performed to identify additional proteins that mediate ciliary trafficking of Amo. Drosophila homologues of KPC1 and KPC2, which comprise the mammalian KIP1 ubiquitination-promoting complex (KPC), form a conserved unit that is required for the sperm tail tip localization of Amo. Male flies lacking either KPC1 or KPC2 phenocopy amo mutants and are sterile due to a failure of sperm storage. KPC is a heterodimer composed of KPC1, an E3 ligase, and KPC2 (or UBAC1), an adaptor protein. Like their mammalian counterparts Drosophila KPC1 and KPC2 physically interact and they stabilize one another at the protein level. In flies, KPC2 is monoubiquitinated and phosphorylated and this modified form of the protein is located in mature sperm. Neither KPC1 nor KPC2 directly interact with Amo but they are detected in proximity to Amo at the tip of the sperm flagellum. In summary this study has identified a new complex that is involved in male fertility in Drosophila melanogaster (Li 2020).

Tissue homeostasis and repair relies on proper communication of stem cells and their differentiating daughters with the local tissue microenvironment. In the Drosophila male germline adult stem cell lineage, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small organoid, the functional unit of differentiation. This study show that cell polarity and vesicle trafficking influence signal transduction in cyst cells, with profound effects on the germ cells they enclose. The data suggest that the cortical components Dlg, Scrib, Lgl and the clathrin-mediated endocytic (CME) machinery downregulate epidermal growth factor receptor (EGFR) signaling. Knockdown of dlg, scrib, lgl, or CME components in cyst cells resulted in germ cell death, similar to increased signal transduction via the EGFR, while lowering EGFR or downstream signaling components rescued the defects. This work provides insights into how cell polarity and endocytosis cooperate to regulate signal transduction and sculpt developing tissues (Papagiannouli, 2019).

The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. A genetic screen targeting regulators of mitochondrial dynamics revealed that mitochondrial fusion is required for the maintenance of male germline stem cells (GSCs) in Drosophila melanogaster. Depletion of Mitofusin (dMfn) or Opa1 led to dysfunctional mitochondria, activation of Target of rapamycin (TOR) and a marked accumulation of lipid droplets. Enhancement of lipid utilization by the mitochondria attenuated TOR activation and rescued the loss of GSCs that was caused by inhibition of mitochondrial fusion. Moreover, constitutive activation of the TOR-pathway target and lipogenesis factor Sterol regulatory element binding protein (SREBP) also resulted in GSC loss, whereas inhibition of SREBP rescued GSC loss triggered by depletion of dMfn. These findings highlight a critical role for mitochondrial fusion and lipid homeostasis in GSC maintenance, providing insight into the potential impact of mitochondrial and metabolic diseases on the function of stem and/or germ cells (Senos, 2019).

The Drosophila male stem cell niche is a well characterized structure in which a small cluster of somatic cells send self-renewal signals to neighbouring germ cells. Although the molecular information involved in the stem cell fate have been identified, much less is understand on the mechanisms driving their short-range specific release. Ultrastructural analysis reveals distinct protrusions of the stem cell plasma membrane that interdigitate with membrane protrusions of the facing hub cells. Some of these protrusions are very elongated and extend into the hub and could correspond to the Mt-Nanotubes. Therefore, the interface between the stem cells and the hub appears more complex than previously reported and the membrane protrusions of the stem cells might represent specialized surface areas involved in the niche-stem cell communication. The presence was noticed of clathrin-coated vesicles in the germline plasma membrane that might be also involved in delivering information from the hub (Persico, 2019).

Hub cells comprise a niche for germline stem cells and cyst stem cells in the Drosophila testis. Hub cells arise from common somatic gonadal precursors in embryos, but the mechanism of their specification is still poorly understood. This study found that RNA binding proteins Lin28 and Imp mediate transcript stability of Bowl, a known hub specification factor; Bowl transcripts were reduced in the testis of Lin28 and Imp mutants, and also when RNA-mediated interference against Lin28 or Imp was expressed in hub cells. In tissue culture Luciferase assays involving the Bowl 3'UTR, stability of Luc reporter transcripts depended on the Bowl 3'UTR and required Lin28 and Imp. These findings suggest that proper Bowl function during hub cell specification requires Lin28 and Imp in the testis hub cells (To, 2021).

The Drosophila male germline stem cell (GSC) lineage provides a great model to understand stem cell maintenance, proliferation, differentiation, and dedifferentiation. This study used Drosophila GSC lineage to systematically analyze transcriptome of discrete but continuous differentiating germline cysts. First single cysts were isolated at each recognizable stage from wild-type testes, which were subsequently applied for RNA-seq analyses. The data delineate a high-resolution transcriptome atlas in the entire male GSC lineage: The most dramatic switch occurs from early to late spermatocyte, followed by the change from the mitotic spermatogonia to early meiotic spermatocyte. By contrast, the transit-amplifying spermatogonia cysts display similar transcriptomes, suggesting common molecular features among these stages, which may underlie their similar behavior during both differentiation and dedifferentiation processes. Finally, distinct differentiating germ cell cyst samples do not exhibit obvious dosage compensation of X-chromosomal genes, even with consideration of the paucity of X-chromosomal gene expression during meiosis, which is different from somatic cells. Together, the single cyst-resolution, genome-wide transcriptional profile analyses provide an unprecedented resource to understand many questions in both germ cell biology and stem cell biology fields (Shi, 2020).

Maintenance of a multicellular organism during homeostasis and tissue repair requires active replenishment of depleted or injured cells during aging and regeneration. Adult stem cells fulfill this requirement owing to their unique ability to both self-renew and give rise to differentiated special cell types. In many adult stem cell lineages, progenitor cells often undergo a proliferative stage to expand their population before commitment for terminal differentiation. The switch from proliferation to differentiation must be tightly regulated, because mis-regulation of this transition might lead to tumorigenesis or tissue dystrophy. On the other hand, progenitor cells remain plastic and can dedifferentiate to become stem cell-like cells in multiple stem cell lineages. In order to properly differentiate adult stem cells or progenitor cells in vitro and/or to promote dedifferentiation in vivo for regenerative medicine, it is necessary to fully understand the molecular changes underlying the normal differentiation program of adult stem cells in vivo (Shi, 2020).

Drosophila spermatogenesis provides a great model system to study mechanisms that regulate the maintenance, proliferation and proper differentiation of adult stem cells. In adult testes of Drosophila melanogaster, germline stem cells (GSCs) can be precisely located by their proximity and attachment to a group of post-mitotic somatic cells called hub cells at the apical tip of testis. A GSC typically divides asymmetrically to self-renew and to give rise to a gonialblast (GB), the daughter cell that initiates differentiation. GBs first go through a transit-amplifying stage as spermatogonial cells, for which they undergo exactly four rounds of mitosis in D. melanogaster. Once this stage is complete, germ cells enter the spermatocyte stage, which is an elongated G2 phase of meiosis I. During this stage, each spermatocyte grows ~25-fold in volume, which involves a robust gene expression program allowing for meiotic divisions and spermatid terminal differentiation program. In parallel with GSC asymmetric cell division, the cyst stem cell (CySC), two of them surrounding each GSC, also divides asymmetrically, resulting in one daughter cell retaining CySC identity and the other daughter cell becoming a differentiated cyst cell. Two cyst cells encapsulate the differentiating germ cells throughout the entire spermatogenesis and they never divide again. It has been demonstrated that CySCs and cyst cells communicate with their accompanying germ cells via multiple signaling pathways for maintaining germ cell fate and regulating proper germline proliferation and differentiation throughout spermatogenesis. During this process, dynamic changes in the gene expression program are orchestrated by both extrinsic cues, such as paracrine factors that trigger signaling pathways, and intrinsic factors, such as chromatin regulators (Shi, 2020).

Previous studies have attempted to parse the transcriptional networks underlying GSC differentiation by comparing gene expression profiles of mutant testes that accumulate germ cells at distinct cellular differentiation stages with wild-type testes. Although these approaches have led to many interesting functional studies using a candidate gene approach, the information gleaned from intact tissues is limited by the inherently mixed population of cells, and the difficulty in extrapolating results obtained from mutant backgrounds to normal situation in wild-type tissue. This report systematically studied the gene expression profile of the Drosophila male GSC lineage at every recognizable and isolatable stage. Using this dataset, A the following questions were addressed: Do GSCs and GBs, the two daughter cells derived from GSC asymmetric division, have similar or distinct transcriptional profiles? How does the transcriptome change in continuously proliferating spermatogonial cells? Is the switch from mitosis to meiosis accompanied by a transcriptome change that leads to another transcriptome change during spermatocyte maturation? Does dosage compensation occur in germ cells? In summary, the single-cyst transcriptome profiles provide a comprehensive dataset at a resolution that has not been achieved before, which yields much-needed information on transcriptional status at each crucial stage from an endogenous stem cell system. Researchers from both germ cell biology and stem cell biology fields should benefit from using this resource to screen for genes with a particular expression pattern or examine genes from specific pathway(s), before designing detailed functional analyses (Shi, 2020).

The single cyst-resolution, genome-wide transcriptional profile analyses provide a data resource at each crucial stage of differentiation in an adult stem cell system under their wild-type situation, which will help in understanding many interesting issues in stem cell and germ cell biology fields. Previously, RNA-seq analyses were carried out using the entire testes with genetic mutations that arrest cellular differentiation pathways at distinct stages or dissected different regions of wild-type testes enriched with distinct stages of germ cells. However, caveats arise from mixed cell types within either intact or dissected tissues, as well as complications using genetic mutations. Furthermore, although single cell RNA-seq could be currently carried out using dissociated single cells, the relatively small number of early-stage cells, including germline stem cells, make it very challenging to obtain their transcriptomes. Single post-meiotic cysts have been isolated and applied for quantitative RT-PCR, which has revealed two dozen transcribed genes in spermatids. In this study, for the TASC experiments, single germline cysts were isolated from niche to late spermatocyte stages using cell type- and stage-specific markers, which allowed precise reconstruction of the single-cyst transcriptome atlas during the entire spermatogenesis process. Therefore, both the original technologies that were developed and the new knowledge gained in this study represent technical and conceptual advances, which will have a broad and long-term impact on studies in the stem cell and germ cell biology fields (Shi, 2020).

As a proof of principle, several new discoveries have already been made using the TASC dataset. For example, a candidate gene called slamdance (sda) was identified that has enriched transcription in the niche sample. The sda gene encodes an aminopeptidase, the function of which had never been reported in any adult stem cell systems. Extensive functional analyses was carried out and it was demonstrated that the Sda protein indeed acts as an aminopeptidase, the in vivo function of which depends on its enzymatic domain. Sda plays important roles in regulating GSC maintenance and progenitor germline dedifferentiation. In addition, it was found that the known germline differentiation factor bam has prolonged transcription in early-stage spermatocytes , even though Bam protein is downregulated. This difference between mRNA expression pattern and protein enrichment indicated a potential post-transcriptional regulation of the bam transcript, which was revealed later through recognition of the 3' untranslated region of bam by microRNAs. Therefore, the TASC dataset can be used to identify genes with interesting expression patterns for in-depth functional analysis, or combined with other data such as protein expression patterns for studying post-transcriptional regulation in the germline, etc. Together, these studies will contribute to better understanding of gene regulation during spermatogenesis, which will also significantly enhance knowledge of reproductive biology (Shi, 2020).

In summary, this single cyst-resolution, genome-wide transcriptional profile analysis provides a supreme and unprecedented data resource at each crucial stage of differentiation in an adult stem cell system under its physiological conditions, which will help in understanding many issues in germ cell biology and stem cell biology fields. The new technologies that were developed and the knowledge gained in this study will have broad and high impact on basic research as well as on regenerative medicine (Shi, 2020).

Adult stem cells divide to renew the stem cell pool and replenish specialized cells that are lost due to death or usage. However, little is known about the mechanisms regulating how stem cells adjust to a demand for specialized cells. A failure of the stem cells to respond to this demand can have serious consequences, such as tissue loss, or prolonged recovery post injury. This study challenged the male germline stem cells (GSCs) of Drosophila melanogaster for the production of specialized cells, sperm cells, using mating experiments. Repeated mating reduced the sperm pool and increased the percentage of GSCs in M- and S-phase of the cell cycle. The increase in dividing GSCs depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNA-i) constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males. Consistent with a role for the G-proteins in regulating GSC division frequency, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to increase the numbers of dividing GSCs in response to mating (Malpe, 2020).

This study shows that repeated mating reduced the sperm pool and increased GSC division frequency. Using highly controlled experiments, it was demonstrated that mated males had more GSCs in M-phase and S-phase of the cell cycle compared to non-mated males. Mated males also showed faster incorporation of EdU in feeding experiments, suggesting that the GSCs of mated males entered the S-phase of the cell cycle more frequently. Though the possibility that mated males ingested more EdU-supplemented food than their non-mated siblings cannot be excluded, the data suggest that the GSCs in mated males cycle faster. The response curve obtained in a time-course experiment is different from the response curves reported by other groups that used bromo-deoxy-uridine (BrDU) as the thymidine analog instead of EdU. For example, the non-mated males in the current experiment had about 70% of EdU-positive GSCs after 48 hours of feeding. A study that uses white (w) mutant animals and fed the same concentration of the thymidine homologue had a steeper response curve, in which 85% of the GSCs were BrDU-marked after 48 hours of feeding. Another study using y, v flies showed even steeper response curves where 100% of the GSCs were BrDU-labeled after 24 hours. However, in this study, animals were fed a 30 times higher concentration of the thymidine homologue than used in the current study. It is proposed that the different response curves are due to the different genetic backgrounds, chemicals, and doses (Malpe, 2020).

These findings demonstrate that GSCs can respond to a demand for sperm by increasing their mitotic activity. Based on RNA-i targeting G-proteins and a dominant negative construct against Gγ1, the increase in MIGSC of mated males is dependent on G-protein signaling. Furthermore, signal transducers predicted to act downstream of G-proteins and GPCRs predicted to act upstream of G-proteins also appeared to be required for the response to mating. Whether G-protein signaling directly affects GSC division frequency, or whether G-protein-dependent communication among the early stage germline cells impacts the MIGSC remains to be investigated (Malpe, 2020).

Due to the lack of mutants and a potential interference of whole animal knock-down in the behavior of the flies, tissue-specific expression of RNA-i-constructs was used. It is surprising that these studies revealed potential roles for seven instead of a single GPCR in the increase of MIGSC in response to mating. A possible explanation is that some of the RNA-i-lines have off-target effects. RNA-i-hairpins can cause the down-regulation of unintended targets due to stretches of sequence homologies, especially when long hairpins are used. However, with the exception of the RNA-i-line directed against 5-HT7, all lines that produced a phenotype contain second generation vectors with a short, 21 nucleotide hairpin predicted to have no off-target effects. Thus, it is hypothesized that multiple GPCRs regulate the increase in MIGSC in response to mating. Consistent with this, expression of other RNA-i-lines directed against Mth or 5-HT1A interfered with the increase in MIGSC in mated males (Malpe, 2020).

The finding that RNA-i against several GPCRs blocked the increase in MIGSC in mated males suggests a high level of complexity in the regulation of GSC division frequency. One simple explanation could be that the increase in GSC division frequency is dependent on ideal physiological conditions and that lack of any of the seven GPCRs somehow impairs the cell's normal metabolism. Alternatively, the GPCRs could act in concert to impact the MIGSC. In the literature, increasing evidence has emerged that GPCRs can form dimers and oligomers and that these have a variety of functional roles, ranging from GPCR trafficking to modification of G-protein mediated signaling. In C. elegans, two Octopamine receptors, SER-3 and SER-6, additively regulate the same signal transducers for food-deprived-mediated signaling. One possible explanation for the non-redundant function of the two receptors was the idea that they form a functional dimer. In mammalian cells, 5-HT receptors can form homo-dimers and hetero-dimers and, dependent on this, have different effects on G-protein signaling. In cultured fibroblast cells, for example, G-protein coupling is more efficient when both receptors within a 5-HT4 homo-dimer bind to agonist instead of only one. In cultured hippocampal neurons, hetero-dimerization of 5-HT1A with 5-HT7 reduces G-protein activation and decreases the opening of a potassium channel compared to 5-HT1A homo-dimers59. The formation of hetero-dimers of GPCRs with other types of receptors plays a role in depression and in the response to hallucinogens in rodents (Malpe, 2020).

Alternatively, or in addition to the possibility that some or all of the seven GPCRs form physical complexes, a role for several distinct GPCRs in regulating GSC division frequency could be explained by cross-talk among the downstream signaling cascades. One signaling cascade could, for example, lead to the expression of a kinase that is activated by another cascade. Similarly, one signaling cascade could open an ion channel necessary for the activity of a protein within another cascade. Unfortunately, the literature provides little information on Drosophila GPCR signal transduction cascades and only very few mutants have been identified that affect a process downstream of GPCR stimulation. Thus, it remains to be explored how stimulation of the GPCRs and G-proteins increase GSC divisions (Malpe, 2020).

The role for G-protein signaling in regulating the frequency of GSC divisions is novel. The data suggest that the increase in MIGSC in response to mating is regulated by external signals, potentially arising from the nervous system, that stimulate G-protein signaling in the germline. Based on the nature of the GPCRs, the activating signal could be Serotonin, the Mth ligand, Stunted, Octopamine, or two other, yet unknown, signals that activate Mth-l5, and CG12290. It will be interesting to address which of these ligands are sufficient to increase MIGSC, in what concentrations they act, by which tissues they are released, and whether they also affect other stem cell populations (Malpe, 2020).

Stem cell activity and cell differentiation is robustly influenced by the nutrient availability in the gonads. The signal that connects nutrient availability to gonadal stem cell activity remains largely unknown. This study shows that tumor necrosis factor Eiger (Egr) is upregulated in testicular smooth muscles as a response to prolonged protein starvation in Drosophila testis. While Egr is not essential for starvation-induced changes in germline and somatic stem cell numbers, Egr and its receptor Grindelwald influence the recovery dynamics of somatic cyst stem cells (CySCs) upon protein refeeding. Moreover, Egr is also involved in the refeeding-induced, ectopic expression of the CySC self-renewal protein and the accumulation of early germ cells. Egr primarily acts through the Jun N-terminal kinase (JNK) signaling in Drosophila. This study shows that inhibition of JNK signaling in cyst cells suppresses the refeeding-induced abnormality in both somatic and germ cells. In conclusion, this study reveals both beneficial and detrimental effects of Egr upregulation in the recovery of stem cells and spermatogenesis from prolonged protein starvation (Chang, 2020).

The stem cell niche regulates the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previous work has identified a series of genes encoding ribosomal proteins that may contribute to the self-renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood. Flies were used to generate tissue-specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT-PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern. Using a genetic manipulation approach, this study investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. RpS13 was shown to be required for the self-renewal and differentiation of GSCs. RpS13 regulates cell proliferation and apoptosis. Mechanistically, RpS13 regulates the expression of ribosome subunits and could moderate the expression of the Rho1, DE-cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE-cad and Arm proteins. These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis (Wang, 2020). CG6015 controls spermatogonia transit-amplifying divisions by epidermal growth factor receptor signaling in Drosophila testes

Spermatogonia transit-amplifying (TA) divisions are crucial for the differentiation of germline stem cell daughters. However, the underlying mechanism is largely unknown. The present study demonstrated that CG6015 was essential for spermatogonia TA-divisions and elongated spermatozoon development in Drosophila melanogaster. Spermatogonia deficient in CG6015 inhibited germline differentiation leading to the accumulation of undifferentiated cell populations. Transcriptome profiling using RNA sequencing indicated that CG6015 was involved in spermatogenesis, spermatid differentiation, and metabolic processes. Gene Set Enrichment Analysis (GSEA) revealed the relationship between CG6015 and the epidermal growth factor receptor (EGFR) signaling pathway. Unexpectedly, it was discovered that phosphorylated extracellular regulated kinase (dpERK) signals were activated in germline stem cell (GSC)-like cells after reduction of CG6015 in spermatogonia. Moreover, Downstream of raf1 (Dsor1), a key downstream target of EGFR, mimicked the phenotype of CG6015, and germline dpERK signals were activated in spermatogonia of Dsor1 RNAi testes. Together, these findings revealed a potential regulatory mechanism of CG6015 via EGFR signaling during spermatogonia TA-divisions in Drosophila testes (Yu, 2021).

Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. Previous work has shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. This study demonstrates that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, these results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception (Ladyzhets, 2020).

The homeostasis of the stem cell niche is regulated by both intrinsic and extrinsic factors, and the complex and ordered molecular and cellular regulatory mechanisms need to be further explored. In Drosophila testis, germline stem cells (GSCs) rely on hub cells for self-renewal and physical attachment. GSCs are also in contact with somatic cyst stem cells (CySCs). Utilizing genetic manipulation in Drosophila, this study investigated the role of Wnt6 in vivo and in vitro. In Drosophila testis, Wnt6 was found to be required for GSC differentiation and CySC self-renewal. In Schneider 2 (S2) cells, Wnt6 was found to regulate cell proliferation and apoptosis. Mechanistically, it was demonstrated that Wnt6 can downregulate the expression levels of Arm, Rac1 and Cdc42 in S2 cells. Notably, Rac1 and Cdc42, which act downstream of the noncanonical Wnt signalling pathway, imitated the phenotypes of Wnt6 in Drosophila testis. Thus, the newly discovered Wnt6-Rac1/Cdc42 signal axis is required for the homeostasis of the stem cell niche in the Drosophila testis (Wang, 2021).

The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. This study presents a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identifies genes required for its encoding in offspring. Intriguingly, as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. Requirements were identified for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, evidence is found that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for understanding of phenotypic variation and evolution (Ost, 2014).

This study shows that acute dietary interventions, as short as 24 hr, have the capacity to modify F1 offspring phenotype via the male germline. Reprogramming occurs in response to dietary manipulations over a physiological range, and phenotypic outcomes require polycomb- and H3K9me3-centric plasticity in spatially and chromatin-state-defined regions of the genome. The eye color shifts in w^{m4 h} offspring and the reduced fat body H3K9me3 staining in adult IGMR offspring supports the conclusions, first, that there are chromatin state changes and, second, that these are stable lifelong. These data are corroborated by selective derepression of Su(var)3-9, SETBD1, Su(var)4-20, and polycomb-sensitive transcripts; chromatin-state-associated transcriptional rearrangements genome wide; selective reprogramming of highly dynamic histone-mark-defined regions; and the fact that intergenerational metabolic reprogramming (IGMR ) itself is sensitive to a string of distinct H3K9me3-centric and polycomb mutants. Although nontrivial, ChIP-seq comparisons of repressive chromatin architecture in mature sperm and multiple defined offspring tissues will be important to establishing the ubiquitousness of these regulatory events and the nature of intergenerational signal itself. These data highlight how acutely sensitive intergenerational control can be to even normal physiological changes, and they identify some of the first genes absolutely required for transmission evolution (Ost, 2014).

First categorized simply as heterochromatin versus euchromatin, multiple empirical models now divide the genome into 5 to 51 chromatin states, depending on the analysis. Paternal high sugar increases gene expression preferentially of heterochromatic-embedded genes in embryos. Specifically, these genes are characterized by active deposition of H3K9me3 and H3K27me3, by long distance from class I insulators, and by sensitivity to fully intact expression of Su(var)3-9, Su(var)4-20, SetDB1, Pc, and E(z) . The data support a model where phenotype has been evolutionarily encoded directly into the chromatin state of relevant loci. Specifically, an abundance of genes important to both cytosolic and mitochondrial metabolism appear to be embedded into H3K9me3- and distinct polycomb-dependent control regions. Indeed, GO analysis of the five chromatin colors indicate a largely mutually exclusive picture, in which functional pathways are not randomly distributed across chromatin states. The paternal IGMR data set revealed clear and strong overlaps with pathways of black (lamin-associated) and blue (polycomb) chromatin and included many key metabolic pathways, including glycolysis, TCA cycle, mitochondrial OxPhos, chitin, and polysaccharide metabolism, changes that could well prime the system for altered functionality given the appropriate stimulus. Indeed, paternal IGMR phenotype is a susceptibility to diet-induced obesity and is most readily observable upon high-sugar diet challenge evolution (Ost, 2014).

The data support a trans-acting mechanism. In the w^m4h experiments, male offspring inherited their X chromosome and thus the reporter from their unchallenged mothers, i.e., the reporter allele never encounters the initial signal but is reproducibly reprogrammed. Further, the failure of Su(var) 4-20^SP and SetDB1¹⁴⁷³ mutants to elicit IGMR responses in their wild-type offspring indicate that wild-type haploid sperm carry the same insufficient reprogramming template as their syncytial mutant counterparts. cis- and trans-acting mechanisms are not mutually exclusive though. Signals transmitted via paternal chromosomes, though likely transmitted in cis, may be manifest via expression of paternal transcripts, which then act in trans. Paternal reductions of Su(var)3-9, SetDB1, and Hp1, for instance, would affect the maternal genome in trans evolution (Ost, 2014).

Despite their genetic similarity, isogenic or congenic animals reared under controlled conditions exhibit measurable variation in essentially all phenotypes. Such variability in genome output is thought to arise largely from probabilistic or chance developmental events in early. This study mapped a mechanism that couples acute paternal feeding and zygotic chromatin state integrity directly to phenotypic output of the next generation. These same signatures predict obesity susceptibility in isogenic mouse and human obesity cohorts. Because acute circadian fluctuations in feeding are essentially constant over evolutionary timescales, they are the perfect mechanistic input upon which a system could evolve to ensure defined phenotypic variation within a given population evolution (Ost, 2014).

Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. This study shows that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo (Levine, 2015).

Faithful chromosome segregation requires careful orchestration of chromosomal condensation, alignment, and movement of mitotic chromosomes during every eukaryotic cell division. The very first embryonic mitosis in animals requires additional synchronization. Paternally and maternally inherited genomes undergo independent chromatin reorganization and replication prior to mitotic entry. For instance, maternal chromosomes must complete meiosis and then transition from a meiotic conformation to an interphase-like state in preparation for replication. The sperm-deposited, paternal chromosomes must undergo an even more radical transition from a highly compact, protamine-rich state to a decondensed, histone-rich state before DNA replication. Despite these divergent requirements to achieve replication- and mitotic-competency, maternal and paternal genomes synchronously enter the first mitosis. Failure to carry out paternal chromosome remodeling in a timely fashion results in paternal genome loss and embryonic inviability (Levine, 2015).

The transition from a protamine-rich sperm nucleus to a competent paternal pronucleus requires the action of numerous maternally deposited proteins in the egg. For instance, paternal genome decondensation post-fertilization requires the integration of histone H3.3, a histone variant deposited by the maternal proteins HIRA, CHD1, and Yemanuclein. Similarly, maternally-deposited MH/Spartan protein localizes exclusively to the replicating paternal genome and is required for faithful paternal chromosome segregation during the first embryonic division. These and other studies demonstrate the essential role of maternally-deposited machinery in rendering competent sperm-deposited DNA and ultimately, ensuring faithful paternal genome inheritance (Levine, 2015).

Is paternal control also necessary for the extensive decondensation and re-condensation of the post-fertilization paternal genome? If so, disruption of such control would manifest as paternal effect lethality (PEL). Unlike male sterility mutants that lack motile sperm, PEL mutants make abundant motile sperm that fertilize eggs efficiently. However, embryos 'fathered' by PEL mutants are inviable. Only a handful of PEL genes have been characterized in animals. These encode proteins that mediate sperm release of paternal DNA, sperm centriole inheritance, and paternal chromosome segregation. Only one of these PEL proteins directly localizes to paternal chromosomes; the sperm-inherited K81 protein localizes exclusively to paternal chromosome termini and ensures telomere integrity. The maintenance of telomeric epigenetic identity joins a growing list of examples of sperm-to-embryo information transmission via protein or RNA inheritance (e.g., diet, stress, embryonic patterning, transcriptional competency). Despite a new appreciation of paternal control over epigenetic information transfer, there are no reports of paternal control over the global chromatin reorganization required for synchronous mitosis across paternally and maternally inherited genomes. Indeed, in the absence of any known paternal protein-directed genome remodeling, a model has emerged that maternal proteins might be sufficient for transforming tightly packaged sperm DNA into a fully competent paternal pronucleus (Levine, 2015).

The notion that maternal control is sufficient to accomplish paternal genome remodeling is challenged by recent findings from the intracellular Wolbachia bacterium that infects more than 50% of insect species. Wolbachia-infected Drosophila males mated to uninfected females father embryos that arrest soon after the first zygotic mitosis. Embryonic arrest occurs because paternal genomes enter the first mitosis with unresolved sister chromatids that fail to separate on the mitotic spindle. Although the identity of the host factor(s) manipulated by Wolbachia to mediate this transgenerational effect is still unknown, what is clear is that pre-fertilization, Wolbachia subverts the paternal germline machinery that helps direct global genome remodeling of paternal chromosomes in the embryo. Wolbachia action during spermiogenesis leads to paternal-maternal genome asynchrony and ultimately, failure of paternal chromosomes to separate on the first mitotic spindle. Despite decades of interest, the molecular basis of paternal control has remained elusive (Levine, 2015).

To investigate the potential for paternal control over sperm genome remodeling post-fertilization, a candidate gene approach was taken, focusing on the Heterochromatin Protein 1 (HP1) proteins that orchestrate genome-wide chromosomal organization in plants, animals, fungi, and some protists. HP1 proteins are defined as such by a combination of two domains - a chromodomain that mediates protein-histone interactions and a chromoshadow domain that mediates protein-protein interactions. The biochemical properties of HP1 members support a diversity of chromatin-dependent processes in the soma, including DNA replication, telomere integrity, and chromosome condensation (Levine, 2015).

Recently, a detailed phylogenomic analysis of the HP1 gene family was carried out in Drosophila that revealed numerous testis-restricted HP1 proteins. Given the established roles of HP1 proteins, it was posited that these newly discovered male-specific HP1 genes might represent excellent candidates for encoding chromatin functions specialized for paternal genome organization and remodeling in the early embryo. Using detailed genetic and cytological analyses, this study shows that one of these testis-specific HP1 proteins, Heterochromatin Protein 1E (HP1E), is essential for priming the paternal genome to enter embryonic mitosis in synchrony with the maternal genome in D. melanogaster. Intriguingly, HP1E is able to mediate this priming function transgenerationally i.e., the HP1E protein itself is not epigenetically inherited. It was further shown that absence of HP1E especially imperils mitotic fidelity of the heterochromatin-rich, paternal sex chromosomes. Thus, this study firmly establishes that both maternal and paternal control are necessary for paternal genome remodeling in the early Drosophila embryo (Levine, 2015).

Properly coordinated chromosome segregation during virtually all mitotic divisions relies on the function of multiple cell cycle checkpoint proteins. No such cell cycle checkpoint proteins have been identified to act in the very first embryonic mitotic cycle, which must nevertheless accomplish the difficult task of synchronizing maternal and paternal chromosomes that were inherited in very different chromatin states. To investigate the paternal contributions that ensure timely participation of the paternal genome in early embryogenesis, a detailed functional analysis was carried out of the testis-restricted HP1E gene in D. melanogaster. It was found that HP1E encodes a novel function that ensures paternal genome stability in the embryo. Cytological and transcriptome analysis revealed that HP1E is developmentally restricted within the male germline, where it contributes to heterochromatin integrity. HP1E depletion during sperm development results in a highly penetrant PEL phenotype in which paternal chromosomes, especially the paternal sex chromosomes, fail to condense in synchrony with the maternal chromosomes and ultimately cause mitotic catastrophe. It was further shown that the PEL embryonic phenotype could not be rescued by egg-supplied HP1E but could be rescued if the paternal DNA was excluded from participating in embryonic mitosis. These observations support a model under which HP1E acts pre-fertilization to ensure proper chromosome condensation and segregation of paternal chromosomes post-fertilization (Levine, 2015).

The 'hit and run' priming function clearly distinguishes HP1E from all other previously characterized paternal effect lethal genes, which encode proteins that are transmitted to the embryo via sperm. These include the Drosophila paternal chromatin-associated PEL, k81, which encodes a protein that persists on paternal telomeres from late spermatogenesis to the first embryonic mitosis. The HP1E-depletion phenotype is instead reminiscent of Drosophila fathers infected with Wolbachia bacteria crossed to uninfected females. Embryonic lethality induced by Wolbachia testis infection is also caused by a pre-fertilization modification to the paternal genome that results in paternal-maternal chromatin asynchrony and mis-segregation at the very first zygotic mitosis. However, Wolbachia-associated PEL results in mis-segregation of the entire paternal genome rather than just the heterochromatin-rich chromosomes observed in HP1E PEL . Moreover, the HP1E PEL defect is completely independent of Wolbachia (PEL phenotype persists for Wolbachia-free males and females). It is therefore concluded that HP1E supports a novel chromatin requirement to prime paternally inherited genomes for synchronous and successful embryonic mitosis (Levine, 2015).

How does HP1E ensure timely mitotic entry? It is formally possible that the PEL phenotype is the consequence of a dysregulated spermatid transcriptome that is, up- or down-regulation of a downstream gene. However, the finding that HP1E depletion results in the global up-regulation of heterochromatin-embedded genes, together with the observation that the heterochromatin-rich paternal sex chromosomes are most vulnerable to HP1E depletion, lead to favoring the alternate model that HP1E functions as a canonical HP1 protein during spermiogenesis. Based on antibody localization and chromatin bridge morphology, no evidence was found for defects in kinetochore assembly or replication machinery engagement in PEL embryos. Instead, the observation that the lethality phenotype first manifests as decondensed paternal chromosomes relative to maternal chromosomes implicates condensation delay of the heterochromatin-rich sex chromosomes. This delay could be the consequence of incomplete replication. Indeed, large stretches of uninterrupted heterochromatic DNA, as found on the Drosophila sex chromosomes, pose a unique challenge to replication. Alternatively, the mitotic delay may be the result of inadequate condensin protein recruitment, which is required for timely resolution of sister chromatids post-replication. Previous studies have shown that heterochromatin can also impair chromosome condensation. Timely completion of replication and condensation requires the action of HP1E's closest relative, HP1A, in somatic cells. However, in developing spermatids, HP1A localizes to telomeres rather than broadly to heterochromatin as observed in virtually all other cell types. It is posited that HP1E adopts a global, HP1A-like chromatin function during this highly specialized developmental stage and ensures the recruitment or retention of either replication or condensin proteins that are required post-fertilization (Levine, 2015).

Previous studies have shown that HP1A is essential for embryo viability. This study shows that paternally-acting HP1E is also essential for embryogenesis. Both HP1A and HP1E evolve under purifying selection. However, unlike HP1A (encoded by Su(var)205), HP1E has an unusually dynamic evolutionary history. Despite ancient origins, HP1E has been recurrently lost over evolutionary time. HP1E has been apparently replaced by younger, testis restricted HP1 paralogs on at least two occasions during Drosophila evolution. Curiously, Drosophila pseudoobscura and related species encode neither HP1E nor a putative replacement testis-specific HP1 gene. How is the paradox of HP1E essentiality in D. melanogaster reconciled with its loss in D. pseudoobscura? It was previously found that HP1E loss along in D. pseudoobscura-related species occurred during the same 7-million evolutionary period as a major sex chromosome rearrangement event, in which the ancestral Y was lost, a neo-Y chromosome was born, and the ancestral X fused to an autosome. The finding that the D. melanogaster sex chromosomes are especially vulnerable to HP1E depletion, combined with the emergence of novel sex chromosome arrangements along the same narrow branch as HP1E pseudogenization, suggests a model under which rearrangements of heterochromatin-rich sex chromosomes in the obscuragroup rendered HP1E non-essential. Such karyotypic changes can bring distal heterochromatin into closer proximity to euchromatin and be sufficient to alter heterochromatin packaging, replication timing or even delete blocks of satellite repeats. Thus, heterochromatin evolution via chromosomal rearrangements may have obviated maintenance of HP1E's essential heterochromatin function, leading to its degeneration in D. pseudoobscura (Levine, 2015).

The finding that HP1E is essential in D. melanogaster yet lost in the obscura group highlights the lineage-restricted essential requirements of chromatin genes. Intriguingly, the only other characterized PEL gene that supports paternal chromatin function in Drosophila embryos, k81, is similarly lineage-restricted despite being essential for paternal telomere function. In contrast, maternally deposited proteins required for paternal chromatin reorganization following fertilization are generally conserved from fly to human. This dichotomy is striking. It specifically suggests that even though the essential functions of paternal control of DNA deposition and chromatin remodeling for embryonic mitosis are likely to be conserved in most animals, whereas the identity of those genes is not. PEL chromatin genes like HP1E and k81 thus challenge the dogma that ancient, conserved genes always encode essential conserved functions. Not only can young genes rapidly acquire essential chromatin functions due to dynamic chromatin evolution, but chromatin changes, such as those driven by karyotype evolution, may also drive the extinction of ancient genes encoding once-essential functions (Levine, 2015).

Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. This study shows developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).

Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation. Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Upd, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).

The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G1 phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G2 phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G2-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).

Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% - 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G2 phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).

By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).

Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).

In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs with two labeled centrosomes were observed, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).

The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).

In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).

Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother- versus daughter centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).

These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast. In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase. The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).

Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. This study shows that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the hub led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control (Levings, 2016).

A long-standing question concerns how stem cells maintain their identity through multiple divisions. It has been reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. This study shows that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity (Xie, 2015).

Epigenetic phenomena are heritable changes in gene expression or function that can persist throughout many cell divisions without alterations in primary DNA sequences. By regulating differential gene expression, epigenetic processes are able to direct cells with identical genomes to become distinct cell types in humans and other multicellular organisms. However, with the exception of DNA methylation, little is known about the molecular pathways leading to epigenetic inheritance (Xie, 2015).

Prior research has shown that epigenetic events play particularly important roles in ensuring both proper maintenance and differentiation of several stem cell populations. Many types of adult stem cells undergo asymmetric cell division to generate a self-renewed stem cell and a daughter cell that will subsequently differentiate. Mis-regulation of this balance leads to many human diseases, ranging from cancer to tissue dystrophy to infertility. However, the mechanisms of stem cell epigenetic memory maintenance as well as how loss of this memory contributes to disease remain unknown (Xie, 2015).

During the asymmetric division of the Drosophila male germline stem cell (GSC), the pre-existing histone 3 (H3) is selectively segregated to the self-renewed GSC daughter cell whereas newly synthesized H3 is enriched in the differentiating daughter cell known as a gonialblast (GB) (Tran, 2012). In contrast, the histone variant H3.3, which is incorporated in a replication-independent manner, does not exhibit such an asymmetric pattern. Furthermore, asymmetric H3 inheritance occurs specifically in asymmetrically dividing GSCs, but not in the symmetrically dividing progenitor cells. These findings demonstrate that global asymmetric H3 histone inheritance possesses both molecular and cellular specificity. The following model is proposed to explain these findings (Xie, 2015).

First, the cellular specificity exhibited by the H3 histone suggests that global asymmetric histone inheritance occurs uniquely in a cell-type (GSC) where the mother cell must divide to produce two daughter cells each with a unique cell fate. Because this asymmetry is not observed in symmetrically dividing GB cells, asymmetric histone inheritance is proposed to be a phenomenon specifically employed by GSCs to establish unique epigenetic identities in each of the two daughter cells. Second, as stated previously, a major difference between H3 and H3.3 is that H3 is incorporated to chromatin during DNA replication, while H3.3 variant is incorporated in a replication-independent manner. Because this asymmetric inheritance mode is specific to H3, a two-step model is proposed to explain asymmetric H3 inheritance: (1) prior to mitosis, pre-existing and newly synthesized H3 are differentially distributed on the two sets of sister chromatids, and (2) during mitosis, the set of sister chromatids containing pre-existing H3 is segregated to GSCs, while the set of sister chromatids enriched with newly synthesized H3 is segregated to the GB that differentiates (Tran, 2012; Tran, 2013; Xie, 2015 and references therein)

This study reports that a mitosis-enriched H3T3P mark acts as a transient landmark that distinguishes sister chromatids with identical genetic code but different epigenetic information, shown as pre-existing H3-GFP and newly synthesized H3-mKO. By distinguishing sister chromatids containing different epigenetic information, H3T3P functions to allow these molecularly distinct sisters to be segregated and inherited differentially to the two daughter cells derived from one asymmetric cell division. The selective segregation of different populations of histones likely allows these two cells to assume distinct fates: self-renewal versus differentiation. Consequently, loss of proper epigenetic inheritance might lead to defects in both GSC maintenance and GB differentiation, suggesting that both cells need this active partitioning process to either 'remember' or 'reset' their molecular properties (Xie, 2015).

The temporal and spatial specificities of H3T3P make it a great candidate to regulate asymmetric sister chromatid segregation. First, H3T3P is only detectable from prophase to metaphase, the window of time during which the mitotic spindle actively tries to attach to chromatids through microtubule-kinetochore interactions. Second, the H3T3P signal is enriched at the peri-centromeric region, where kinetochore components robustly crosstalk with chromatin-associate factors. Third, H3T3 shows a sequential order of phosphorylation, first appearing primarily on sister chromatids enriched with pre-existing H3 and then subsequently appearing on sister chromatids enriched with newly synthesized H3 as the GSC nears metaphase. The distinct temporal patterns shown by H3T3P are unique to GSCs and would allow the mitotic machinery to differentially recognize sister chromatids bearing distinct epigenetic information; an essential step necessary for proper segregation during asymmetric GSC division. Furthermore, the tight temporal control of H3T3 phosphorylation suggests that rather than serving as an inherited epigenetic signature, H3T3P may act as transient signaling mark to allow for the proper partitioning of H3. It is hypothesized that H3T3P needs to be under tight temporal control in order to ensure proper H3 inheritance and germline activity (Xie, 2015).

These studies have shown that H3T3P is indeed subject to stringent temporal controls during mitosis. The H3T3P mark is undetectable during G2 phase. Upon entry to mitosis, sister chromatids enriched with pre-existing H3-GFP histone begin to show H3T3 phosphorylation prior to sister chromatids enriched with newly synthesized H3-mKO. As the cell continues to progress toward metaphase, H3T3P signal begins to appear on sister chromatids enriched with newly synthesized H3-mKO. Such a tight regulation of H3T3P is compromised when levels of H3T3P are altered due to the incorporation of mutant H3T3A or H3T3D. Incorporation of the H3T3A mutant results in a significant decrease in the levels of H3T3P on sister chromatids throughout mitosis, such that neither sister becomes enriched with H3T3P as the GSC progresses toward metaphase. Conversely, incorporation of the H3T3D mutant would result in seemingly elevated levels of H3T3P early in mitosis. Although H3T3A and H3T3D act in different ways, both mutations significantly disrupt the highly regulated temporal patterns associated with H3T3 phosphorylation, the result of which is randomized H3 inheritance patterns and germ cell defects in testes expressing either H3T3A or H3T3D (Xie, 2015).

To further evaluate the extent of H3T3A and H3T3D roles in the segregation of sister chromatids enriched with different populations of H3 during mitosis, all possible segregation patterns were modeled in male GSCs, and these estimates were compared to the experimental results. To simplify the calculations, two important assumptions were made: first, nucleosomal density was assumed to be even throughout the genome. This assumption allows the inference that the overall fluorescent signal contributed by each chromosome is proportional to their respective number of DNA base pairs. Second, by quantifying pre-existing H3-GFP asymmetry in anaphase and telophase GSCs, it was estimated that the establishment of H3-GFP asymmetry is ∼4-fold biased, i.e., 80% on one set of sister chromatids and 20% on the other set of sister chromatids, based on quantification of GFP signal in anaphase and telophase GSCs (Tran, 2012). With these two simplifying assumptions, both GFP and mKO ratios were caculated among all 64 possible combinations. If asymmetry is designed as a greater than 1.5-fold difference in fluorescence intensity, then based on a model of randomized sister chromatid segregation, it is estimated that a symmetric pattern should appear for 53.1% (34/64) of GSC-GB pairs whereas both conventional and inverted asymmetric patterns should occur with equal frequencies and account for 18.7% (12/64) of total GSC-GB pairs. The remaining 9.4% (6/64) of GSC-GB pairs should produce histone inheritance patterns with a 1.45- to 1.55-fold difference in signal intensity (Xie, 2015).

This estimation is close to the experimental data in both H3T3A- and H3T3D-expressing testes. Of the 64 quantified post-mitotic GSC-GB pairs in nos>H3T3A testes, ∼71.9% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 9.4% and 12.5%, respectively, and 6.3% at the borderline. Similarly, of the 57 quantified post-mitotic GSC-GB pairs in nos>H3T3D testes, ∼79.0% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 7.0% and 10.5%, respectively with 3.5% of pairs at the borderline. Some differences between predicted ratios and the experimental data could be due to the simplified assumptions, the limited sensitivity of the measurement, and/or some coordinated chromatid segregation modes that bias the eventual read-out. In summary, comparison between the modeling ratios and the experimental data suggest that loss of the tight control of H3T3 phosphorylation in GSCs randomizes segregation of sister chromatids enriched with different populations of H3 (Xie, 2015).

If the temporal separation in the phosphorylation of H3T3 on epigenetically distinct sister chromatids facilitates their proper segregation and inheritance during asymmetric cell division, it is likely that mutations of the Haspin kinase will also affect the temporal control of H3T3 phosphorylation. In the context of H3T3A, where the levels of H3T3P are already reduced, a further decrease in H3T3P by reducing Haspin levels should limit the GSC's ability to distinguish between sister chromatids enriched with distinct H3. Indeed, haspin mutants enhance the phenotypes in nos>H3T3A testes. A different situation appears in the context of H3T3D where sister chromatids experience seemingly elevated levels of H3T3P at the start of mitosis. These elevated H3T3P levels may be exacerbated by the phosphorylation activity of the Haspin kinase. Therefore, it is conceivable that by halving the levels of the Haspin kinase, H3T3 phosphorylation should be reduced to a level more closely resembling wild-type. In this way, some of the temporal specificity that is lost in the H3T3D mutant is restored, resulting in suppression of the phenotypes observed in nos>H3T3D testes. An exciting topic for future study would be to further explore how exactly Haspin phosphorylates H3T3 in the context of chromatin and whether H3T3A and H3T3D mutations act synergistically or antagonistically in regulating asymmetric sister chromatids segregation through differential phosphorylation of a key histone residue (Xie, 2015).

It would also be interesting to understand the potential connection between asymmetric histone inheritance and another phenomenon reported by several investigators: selective DNA strand segregation. Recent development of the chromosome orientation fluorescence in situ hybridization (CO-FISH) technique allows study of selective chromatid segregation at single-chromosome resolution. Using this technique in mouse satellite cells, it has been demonstrated that all chromosomes are segregated in a biased manner, such that pre-existing template DNA strands are preferentially retained in the daughter cell that retains stem cell identity. Interestingly, this biased segregation becomes randomized in progenitor non-stem cells. Using CO-FISH in Drosophila male GSCs, sex chromosomes have been shown to segregate in a biased manner. Remarkably, sister chromatids from homologous autosomes have been shown to co-segregate independent of any specific strand preference. Such findings hint at a possible epigenetic source guiding the coordinated inheritance of Drosophila homologous autosomes. In many cases of biased inheritance, researchers have speculated about the existence of a molecular signature that would allow the cell to recognize and segregate sister chromatids bearing differential epigenetic information. However, the identity of such a signature has remained elusive. The work represented in this paper provides experimental evidence demonstrating that a tightly-controlled histone modification, H3T3P, is able to distinguish sister chromatids and coordinate their segregation (Xie, 2015).

Epigenetic processes play important roles in regulating stem cell identity and activity. Failure to appropriately regulate epigenetic information may lead to abnormalities in stem cell behaviors, which underlie early progress toward diseases such as cancer and tissue degeneration. Due to the crucial role that such processes play in regulating cell identity and behavior, the field has long sought to understand whether and how stem cells maintain their epigenetic memory through many cell divisions. Yhe results of this study suggest that the asymmetric segregation of pre-existing and newly synthesized H3-enriched chromosomes may function to determine distinct cell fates of GSCs versus differentiating daughter cells (Xie, 2015).

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). Extended live imaging of the Drosophila testis niche was developed, allowing the tracking of individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event termed 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. GSCs were captured undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements (Sheng, 2011).

Live imaging of the Drosophila germline stem cell niche has directly demonstrated many aspects of GSC behavior that were impossible to observe in fixed tissues. Asymmetrically oriented divisions do not necessarily determine asymmetric cell fate, but can occasionally result in the production of two GSCs. This is the primary mechanism by which GSCs are replenished in healthy tissues to compensate for GSC loss. As GSC-daughter pairs are adjacent to the hub and are enriched in the maintenance factor STAT92E, the process of symmetric renewal is probably distinct from de-differentiation of spermatogonia (which are non hub-adherent and express the differentiation factor Bam). The frequency of symmetric renewal increases during GSC recovery after protein starvation, and during GSC regeneration after genetically induced stem cell depletion. In the latter case, where the rate of GSC regeneration is higher, GSCs are concurrently derived from de-differentiating spermatogonia, a process characterized by movement, fragmentation and adhesion to the hub by spermatogonial cells. Together, these data demonstrate that lost GSCs can be regenerated by multiple mechanisms, some or all of which may be similar to events occurring in other stem cell systems (Sheng, 2011).

As changes in stem cell output are observed during regeneration, signaling from support cells or from systemic factors may underlie these effects. Niche-generating cells, transit amplifying daughter cells or even differentiated daughter cells may potentially signal to stem cells and modulate their division output. In the Drosophila testis, GSC maintenance depends on Jak-STAT signaling initiated from the hub, but it is not known whether this same pathway regulates division outcome. As STAT-null GSCs are rapidly lost from the niche, low levels of Jak-STAT signaling due to fluctuations in gene expression may be sufficient to cause GSC loss. In support of this hypothesis, three out of 556 GSCs examined for STAT92E expression had low levels of this protein. However, the mRNA expression pattern of the Jak-STAT pathway ligand Upd is unchanged during de-differentiation, suggesting that genes other than Upd may affect symmetric renewals. BMP signaling, which is required for GSC maintenance, is a good candidate. Combining live imaging with genetic tools for monitoring levels of signaling pathway activation in the Drosophila testis will provide a powerful platform for understanding how cell signaling affects the outcome of stem cell divisions in real time (Sheng, 2011).

The observation that both symmetric renewal and GSC loss occur when the GSC is attached to a daughter cell suggests that there may be a cell cycle-specific gene expression profile that primes the cells for these events to occur during S or early G2 in the cell cycle. It is speculated that the abscission accompanying symmetric renewal is similar to that occurring in GSC-GB pairs, another G2 event. Cell cycle regulation, which is characterized by a short G1 phase and relatively long S phase, maintains pluripotency in many types of cultured stem cells. As GSCs in the Drosophila testis have short G1 phases, and Drosophila GSCs require distinct cell cycle regulators, investigation of cell cycle regulation of Drosophila GSC division outcome may be informative (Sheng, 2011).

It was shown that GSCs in both centrosomin mutants and starved wild-type flies have increased frequencies of symmetric renewal, but surprisingly, there is no corresponding rise in GSC numbers. These results suggest that increased symmetric renewal is counterbalanced by increased GSC loss. Cnn mutant GSC are reported to have abnormal cell morphology and often appear to be detaching from the hub, suggesting an overall maintenance defect. During starvation, lowered insulin signaling results in GSCs loss, and this effect can be rescued by overactivation of insulin signaling. The results indicate that symmetric renewals of GSCs undergoing oriented divisions are the source of new GSCs. Starved flies initially have low insulin signaling, but when returned to normal food for a day have higher insulin signaling. However, this study found that both timepoints exhibited increased symmetric renewals, leading to the idea that activation of insulin signaling does not directly modulate division outcome. Perhaps during starvation, lowered insulin signaling causes GSC loss, which in turn triggers a compensatory increase in symmetric renewal. However, symmetric renewals are not able to fully compensate for the loss, yielding an overall decrease in GSC number. When flies are re-fed and insulin signaling returns to normal, GSCs are no longer rapidly lost, and the same rate of symmetric renewal is now able to increase overall GSC number. Together, these results suggest that the behavior of stem cells within the niche is much more dynamic than previously expected, and indicate that GSC number is controlled by the relative rates of symmetric renewal versus loss, not by the orientation of the division plane (Sheng, 2011).

Why do the majority of Drosophila GSCs undergo asymmetric division if symmetric renewal plus symmetric differentiation produces the same output? As GSCs and CySCs function together within the niche during spermatogenesis, robust division orientation of both populations may enable differentiating germline cells to be generated at a rate that matches cyst cell production. Asymmetric divisions may also prevent clonal expansion of stem cells harboring harmful mutations within the niche, which can compete for niche occupancy. However, clonal expansion may not always be harmful; mammalian niches regularly progress towards mono-clonality with stem cells exhibiting neutral drift dynamics. Perhaps symmetrically renewing divisions are not detrimental to mammalian systems because mammalian niches are not as constrained spatially, and mammalian stem cells are often motile. So far, asymmetric division in Drosophila testes correlates with optimal GSC function, as it becomes less robust with aging. Whether symmetric divisions increase during aging has not been examined, but it might occur because GSCs are thought to be lost more frequently due to decreased maintenance cues. Interestingly, depleting STAT92E from GSCs displaces them from the hub, yet they are not lost from the tissue. Instead, they associate with BMP-producing CySCs, which probably promote GSC renewal. However, GSC division orientation is now randomized; suggesting that their output is composed of symmetric renewals and symmetric differentiation. Furthermore, APC2 mutants that affect centrosome position and E-cadherin mutants that have misoriented divisions still have wild-type GSC numbers. Together, these observations suggest that the Drosophila testis stem cell niche does not require invariant asymmetric GSC division outcomes (Sheng, 2011).

As mammalian stem cells are thought to undergo symmetric renewal in combination with stochastic differentiation, rather than strict asymmetric divisions, GSCs in Drosophila may share more aspects of stem cell behavior with mammalian systems than has been previously assumed. Wild-type GSCs were observed losing niche attachment and directly differentiating, which is consistent with reports that subsets of undifferentiated spermatogonia in the mouse testes can directly differentiate. Although a lost GSC being replaced by a neighboring GSC undergoing symmetric renewal was observed, this was only a single example where these events are coupled together. Thus, stem cell loss and symmetric renewal may occur stochastically in Drosophila GSCs, as in the mouse testis. It was also shown that differentiating spermatogonia revert into GSCs, which is consistent with findings that differentiating spermatogonia can contribute to the stem cell pool during reconstitution of spermatogenesis in the mouse testes. Therefore, this system provides an ideal platform for determining regulators of stem cell loss and replacement in vivo that may also be conserved in mammalian tissues (Sheng, 2011).

Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. This study shows that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. GSCs containing misoriented centrosomes accumulate with age, and these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, it is proposed that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. It was also shown that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia (Cheng, 2008).

GSCs with misoriented centrosomes accumulate as flies age. Since such misoriented GSCs divide less frequently as compared to oriented GSCs, accumulation of misoriented GSCs contributes to the decline in spermatogenesis that occurs with age. Although misoriented GSCs rarely divide, they are not permanently arrested (or senescent) and are correctly oriented when they divide. Whether correction of GSC orientation is an active process that is part of the acquisition of stem cell identity remains to be determined. The low cell cycle activity of misoriented GSCs may also suggest that mechanisms are in place to detect misorientation and induce cell cycle arrest in response to this change, although the underlying mechanisms remain to be identified (Cheng, 2008).

It was also demonstrated that misoriented GSCs originate, at least in part, from dedifferentiation of spermatogonia. Although dedifferentiated GSCs have high frequency (>40%) of centrosome misorientation, they can function as stem cells by resuming the cell cycle, with correctly oriented mitotic spindles just like as constitutive GSCs. GSC numbers do not decrease as quickly as expected from the calculated GSC half-life, suggesting that a mechanism to compensate for the loss of GSCs exists. Since misoriented spindles, or symmetric stem cell division, was rarely observed, it is speculated that dedifferentiation is the major mechanism to replace stem cells over time in the Drosophila male germ line (Cheng, 2008).

A decline in GSC number in older males (day 50) was reported recently (Boyle, 2007) This decrease in stem cell number is likely due to failure of the niche function (via decreased signal from the niche as well as decreased E-cadherin-based attachment between the niche and GSCs. However, the decrease in the production of spermatogonia and testis involution precede the loss of GSCs such that decreasing GSC numbers cannot explain the testis involution that is observed at younger ages (Cheng, 2008).

The present results provide a novel mechanistic link between the control of stem cell polarity and the age-related decline in tissue regenerative capacity. Mechanisms responsible for monitoring stem cell orientation with respect to the niche not only prevent overproliferation of stem cells by ensuring the asymmetric outcome of the stem cell division, but they contribute to the decline in tissue regenerative capacity during aging. Many of the misoriented GSCs originate from the dedifferentiation of spermatogonia, a mechanism thought to be responsible for maintaining the stem cell population over extended periods of time. Therefore, although GSCs produce less progeny over time, the system appears to maximize the number of progeny produced throughout life, while maintaining asymmetric stem cell division (Cheng, 2008).

In summary, it is proposed that the GSCs with misoriented centrosome divide less frequently and that a combination of such a decreased stem cell division and a higher frequency of the GSC misorientation in aged testes leads to a decline in spermatogenesis with age (Cheng, 2008).

The Drosophila CNS and male germline offer two new tractable systems that can be used to study SMN biology in development and stem cells. It also offers a system to study how SMN, a protein associated with neuronal development, could cause SMA. Although SMA is classically a disease of the motor neuron, a severe reduction of SMN protein affects a wide spectrum of cells including stem cells. Consistent with this idea, symptoms in mild forms of SMA (type III or IV) are predominately limited to motor neurons. However, patients with the most severe type (type I), suffer from defects in multiple tissues including congenital heart defects, multiple contractures, bone fractures, respiratory insufficiency, or sensory neuronopathy. Elucidating the differential requirements of SMN in individual cell types, and how their sensitivity to SMN loss can mediate the disease, can contribute to the understanding of the selectivity of SMA (Grice, 2011).

Stem cells can self-renew and generate differentiating daughter cells. It is not known whether these cells maintain their epigenetic information during asymmetric division. Using a dual-color method to differentially label 'old' versus 'new' histones in Drosophila male germline stem cells (GSCs), it was shown that preexisting canonical H3, but not variant H3.3, histones are selectively segregated to the GSC, whereas newly synthesized histones incorporated during DNA replication are enriched in the differentiating daughter cell (see Experimental design and potential results). The asymmetric histone distribution occurs in GSCs but not in symmetrically dividing progenitor cells. Furthermore, if GSCs are genetically manipulated to divide symmetrically, this asymmetric mode is lost. This work suggests that stem cells retain preexisting canonical histones during asymmetric cell divisions, probably as a mechanism to maintain their unique molecular properties (Tran, 2013).

Although all cells in an organism contain the same genetic material, different genes are expressed in specific cell types, allowing them to differentiate along distinct pathways. Epigenetic mechanisms regulate gene expression and maintain a specific cell fate through many cell divisions. Stem cells have the remarkable ability to both self-renew and generate daughter cells that enter differentiation. Epigenetic mechanisms have been reported to regulate stem cell activity in multiple lineages. However, there has been little direct in vivo evidence demonstrating whether stem cells retain their epigenetic information (Tran, 2013).

The Drosophila male GSCs are well characterized in terms of their physiological location, microenvironment (i.e., niche), and cellular structures). Male GSCs can be identified precisely by their distinct anatomical positions and morphological features. A GSC usually divides asymmetrically to produce a self-renewed GSC and a daughter cell gonialblast (GB) that undergoes differentiation. Therefore, GSCs can be examined at single-cell resolution for a direct comparison (Tran, 2013).

In eukaryotes, the basic unit of chromatin called nucleosome contains histone octamer [2×(H3, H4, H2A, H2B)] and DNA wrapping around them. Indeed, histones are one of the major carriers of epigenetic information. To address how histones are distributed during the GSC asymmetric division, a switchable dual-color method was developed to differentially label 'old' versus 'new' histones that uses both spatial (by Gal4; UAS system) and temporal (by heat shock induction) controls to switch labeled histones from green [green fluorescent protein (GFP)] to red [monomeric Kusabira-Orange (mKO)]. Heat shock treatment induces an irreversible DNA recombination to shut down expression of GFP-labeled old histones and initiate expression of mKO-labeled new histones. If the old histones are partitioned nonselectively, the GFP will initially exhibit equal distribution in the GSC and GB, and will be gradually replaced by the mKO. However, if the old histones are preferentially retained in the GSCs to constitute potentially GSC-specific chromatin structure, the GFP will be detected specifically in the GSCs. During DNA replication-dependent canonical histone deposition, histones H3 and H4 are incorporated as a tetramer, and histones H2A and H2B are incorporated as dimers. Therefore, independent transgenic strains were generated for H3 and H2B, respectively. On the other hand, histone variants are incorporated into chromatin in a transcription-coupled but DNA replication-independent manner. Therefore, the histone variant H3.3 was used as a control for canonical histones (Tran, 2013).

To avoid potential complications caused by heat shock-induced DNA recombination on either one or both chromosomes in GSCs, each of the three transgenes (H3, H2B, and H3.3) was integrated as a single copy and analyzed in heterozygous flies. Examination of testes with the transgenes revealed nuclear GFP but little mKO signal before heat shock. After heat shock, mKO signals were detectable. Different GSCs undergo mitosis asynchronously, and an average cell cycle length of GSCs is approximately 12 to 16 hours. Among all GSCs, 75% to 77% are in G₂ phase, 21% are in S phase, fewer than 2% are in mitosis, and G₁-phase GSCs are almost negligible. Moreover, the GSC and GB arising from an asymmetric division remain connected after mitosis by a cellular structure known as the spectrosome, when they undergo the next G₁ and S phases synchronously (Tran, 2013).

To examine the distribution of old versus new histones in GSC and GB after a round of DNA replication-dependent histone deposition, testes were studied 16 to 20 hours after heat shock. In particular, GSC-GB pairs connected by spectrosomes were examined. On the basis of cell cycle length of GSCs, these GSC-GB pairs were from GSCs that switched from histone-GFP to histone-mKO genetic code during their G₂ phase and then underwent the first mitosis followed by G₁, S, and G₂ phase and the second mitosis. Within this time frame, both old histones and new histones were detectable in GSCs at the second G₂ phase because new histones had been synthesized and incorporated during the first S phase. For histone H3, the GFP signal was detected primarily in the GSC but not in the GB. By contrast, the mKO signals were present in both the GSC and the GB, with a relatively higher level in the GB. The asymmetric distribution of histone H3 was specific for GSC divisions, because both the GFP and mKO signals were equally distributed in spermatogonial cells derived from a symmetric division of the GB in the same testis samples. Quantification of fluorescence intensity revealed that the old H3 (GFP-labeled) signal was more enriched in the GSC than in the GB by a factor of ~5.7, whereas new H3 (mKO-labeled) signal was more enriched in the GB than in the GSC by a factor of ~1.6. By contrast, this differential distribution of old versus new histone was not detected for symmetrically dividing spermatogonial cells (Tran, 2013).

In contrast to the asymmetric distribution pattern for the canonical histone H3, the histone variant H3.3 did not show this asymmetry during GSC divisions, by fluorescence images and by quantification. The symmetry of the histone variant H3.3 suggests that the asymmetric mode is specific for canonical histone H3 (Tran, 2013).

Fewer than 2% of all GSCs are undergoing mitosis; thus, all analyses above were based on postmitotic GSC-GB pairs. To further examine the histone segregation pattern during mitosis, a screen was carried out for mitotic GSCs. Indeed, old histones were mainly associated with the chromatids segregated to the GSC side at metaphase, anaphase, and telophase. By contrast, new histones were more enriched at the chromatids segregated to GB side). These results suggest that the sister chromatids preloaded with old histones are preferentially retained in GSCs and that the ones enriched with new histones are partitioned to GBs during GSC mitosis (Tran, 2013).

Next, the histone distribution pattern was examined during the first GSC division by recovering GSCs for 4 to 6 hours after heat shock. An asymmetric distribution pattern was also found in the GSC-GB pairs with the H3 transgene. By contrast, a symmetric distribution pattern was observed for both dividing spermatogonial cells with the H3 transgene and H3.3 during GSC division. Quantification of fluorescence intensity revealed that the old H3-GFP signal was enriched in the GSC by a factor of ~13 relative to the GB, whereas the new H3-mKO signal was enriched in the GB by a factor of ~2.4 relative to the GSC. By contrast, there was no differential distribution of the old versus new histone for the symmetrically dividing spermatogonial cells, or H3.3 during GSC division. Although an asymmetric histone distribution pattern was detected in postmitotic GSC-GB pairs, examination of the mitotic GSC at this stage did not show any asymmetry. These data suggest that the asymmetric segregation mode relies on replication-dependent histone incorporation prior to mitosis. However, the factor of >10 difference of GFP signal between GSC and GB could be contributed by faster turnover of old histones in GBs, probably as a mechanism to reset the chromatin for differentiation. By contrast, the difference of mKO in GSC and GB was less substantial, probably as a result of new histone synthesis in both cells. Furthermore, the H2B transgene showed a similar pattern to H3 after the first GSC division (Tran, 2013).

The consistent asymmetric cell divisions of GSCs could be lost under certain conditions, such as ectopic activation of the key JAK-STAT signaling pathway in the niche. It has been shown that overexpression of the JAK-STAT ligand unpaired (OE-upd) induces overpopulation of GSCs. Consistent with the loss of asymmetry in expanded GSCs, the asymmetric distribution pattern of the histone H3 was not observed in OE-upd testes 16 to 20 hours after heat shock. These results demonstrate that the asymmetric histone distribution pattern is dependent on GSC asymmetric divisions. A two-step process is proposed as the favored explanation: Old and newly synthesized histones are incorporated to different sister chromatids during S phase; then, during mitosis, the sister chromatid preloaded with old histones is preferentially segregated to GSC (Tran, 2013).

These data reveal that stem cells preserve preexisting histones through asymmetric cell divisions. The JAK-STAT signaling pathway required for the asymmetric GSC divisions contributes to the asymmetric histone distribution pattern. This work provides a critical first step toward identifying the detailed molecular mechanisms underlying old histone retention during GSC asymmetric division. These findings in the well-characterized GSC model system will facilitate understanding of how epigenetic information could be maintained by stem cells or reset in their sibling cells that undergo cellular differentiation (Tran, 2013).

Self-renewal and differentiation in germline stem cells (GSCs) are tightly regulated by the stem cell niche and via multiple approaches. In a previous study, the novel GSC regulatory gene Srlp (CG5844) was screened in Drosophila testes. However, the underlying mechanistic links between Srlp and the stem cell niche remain largely undetermined. Using genetic manipulation of the Drosophila model, this study systematically analyzed the function and mechanism of Srlp in vivo and in vitro. In Drosophila, Srlp is an essential gene that regulates the self-renewal and differentiation of GSCs in the testis. In the in vitro assay, Srlp is found to control the proliferation ability and cell death in S2 cells, which is consistent with the phenotype observed in Drosophila testis. Furthermore, results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) reveal that RpL6 binds to Srlp. Srlp also regulates the expression of spliceosome and ribosome subunits and controls spliceosome and ribosome function via RpL6 signals. Collectively, these findings uncover the genetic causes and molecular mechanisms underlying the stem cell niche. This study provides new insights for elucidating the pathogenic mechanism of male sterility and the formation of testicular germ cell tumor (Yu, 2019).

Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. How non-genic information could be inherited differentially to establish distinct cell fates is not well understood. This study reports a series of spatiotemporally regulated asymmetric components, which ensure biased sister chromatid attachment and segregation during ACD of Drosophila male germline stem cells (GSCs). First, sister centromeres are differentially enriched with proteins involved in centromere specification and kinetochore function. Second, temporally asymmetric microtubule activities and polarized nuclear envelope breakdown allow for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. Abolishment of either the asymmetric sister centromeres or the asymmetric microtubule activities results in randomized sister chromatid segregation. Together, these results provide the cellular basis for partitioning epigenetically distinct sister chromatids during stem cell ACDs, which opens new directions to study these mechanisms in other biological contexts (Ranjan, 2019).

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. This study aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, it is proposed that 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, this study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels (Riera-Escamilla, 2022).

Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, this study focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. Single embryo PCR was developed and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, this study found increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI (Warecki, 2022).

Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, this study shows that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. This study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity (Terretaz, 2023).

Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, among which the host modification (HM) model predicts that Wolbachia effectors play an important role in sperm modification. However, owing to the complexity of spermatogenesis and testicular cell-type heterogeneity, whether Wolbachia have different effects on cells at different stages of spermatogenesis or whether these effects are linked with CI remains unknown. Therefore, this study used single-cell RNA sequencing to analyse gene expression profiles in adult male Drosophila testes that were infected or uninfected by Wolbachia. Wolbachia was found to be significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells, resulting in dysregulated expression of genes related to DNA compaction, and Wolbachia infection also influenced the histone-to-protamine transition in the late stage of sperm development. These results support the HM model and suggest that future studies on Wolbachia-induced CI should focus on cells in the early stages of spermatogenesis (Dou, 2023).

Homologous recombination (HR) repair is uniquely regulated in the ericentromeric heterochromatin to enable 'safe' repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. This study induced DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR- white reporter and I-SceI expression in spermatogonia of flies. Using this approach, higher frequency of HR repair is detected in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin (Miller, 2023).

It has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. This study explored the role of UBR4 on fertility with a Drosophila model. Different Ubr4 knockdown flies were established using the UAS/GAL4 activating sequence system. Fertility, hatchability, and testis morphology were studied, and bioinformatics analyses were conducted. The results indicated that UBR4 deficiency could induce male sterility and influent egg hatchability in Drosophila. Ubr4 deficiency affected the testis during morphological analysis. Proteomics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of Ubr4 knockdown flies. Gene Ontology analysis revealed significant upregulation of CG11598 and Sfp65A, and downregulation of Pelota in Ubr4 knockdown flies. These proteins were involved in the biometabolic or reproductive process in Drosophila. These regulated proteins are important in testis generation and sperm storage promotion. Bioinformatics analysis verified that expression of UBR4 was low in cryptorchidism patients, which further supported the important role of UBR4 in male fertility. Overall, these findings suggest that UBR4 deficiency could promote male infertility and may be involved in the protein modification of UBR4 by upregulating Sfp65A and CG11598, whereas downregulating Pelota protein expression (Xie, 2023). The origin and structural evolution of de novo genes in Drosophila

Although previously thought to be unlikely, recent studies have shown that de novo gene origination from previously non-genic sequences is a relatively common mechanism for gene innovation in many species and taxa. These young genes provide a unique set of candidates to study the structural and functional origination of proteins. However, understanding of their protein structures and how these structures originate and evolve are still limited, due to a lack of systematic studies. This study combined high-quality base-level whole genome alignments, bioinformatic analysis, and computational structure modeling to study the origination, evolution, and protein structure of lineage-specific de novo genes. 555 de novo gene candidates were identified in D. melanogaster that originated within the Drosophilinae lineage. A gradual shift was found in sequence composition, evolutionary rates, and expression patterns with their gene ages, which indicates possible gradual shifts or adaptations of their functions. Surprisingly, little overall protein structural changes were found for de novo genes in the Drosophilinae lineage. Using Alphafold2, ESMFold, and molecular dynamics, a number of de novo gene candidates were identified with protein products that are potentially well-folded, many of which are more likely to contain transmembrane and signal proteins compared to other annotated protein-coding genes. Using ancestral sequence reconstruction, it was found that most potentially well-folded proteins are often born folded. Interestingly, one case was observed where disordered ancestral proteins become ordered within a relatively short evolutionary time. Single-cell RNA-seq analysis in testis showed that although most de novo genes are enriched in spermatocytes, several young de novo genes are biased in the early spermatogenesis stage, indicating potentially important but less emphasized roles of early germline cells in the de novo gene origination in testis. This study provides a systematic overview of the origin, evolution, and structural changes of Drosophilinae -specific de novo genes (Peng,2023). Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development

Testis has the largest number of proteins and tissue-specific proteins in animals. Knockdown of ocnus (ocn), a testis-specific gene, has been shown to result in much smaller testis with no germ cells in Drosophila. Through iTRAQ quantitative proteomics sequencing, 606 proteins were identified as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes; 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs) were proteins that affected generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. qRT-PCR confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated ones were also identified. In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways. The differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, these results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation (Zheng, 2023).

Ribosomal DNA (rDNA) loci contain hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss due to intrachromatid recombination between rDNA copies, threatening multigenerational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. This study shows that the rDNA-specific retrotransposon R2 is essential for restorative rDNA CN expansion to maintain rDNA loci in the Drosophila male germline. The depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. Double-stranded DNA breaks created by the R2 endonuclease, a feature of R2's rDNA-specific retrotransposition, initiate the process of rDNA CN recovery, which relies on homology-dependent repair of the DNA break at rDNA copies. This study reveals that an active retrotransposon provides an essential function for its host, contrary to transposable elements' reputation as entirely selfish. These findings suggest that benefiting host fitness can be an effective selective advantage for transposable elements to offset their threat to the host, which may contribute to retrotransposons' widespread success throughout taxa (Nelson, 2023). Chronic exposure to warm temperature causes low sperm abundance and quality in Drosophila melanogaster

Temperature influences male fertility across organisms; however, how suboptimal temperatures affect adult spermatogenesis remains understudied. In a recent study on Drosophila melanogaster oogenesis, a drastic reduction was observed in the fertility of adult males exposed to warm temperature (29s°C). This study showss that males become infertile at 29 °sC because of low sperm abundance and quality. The low sperm abundance at 29 °C does not stem from reduced germline stem cell or spermatid numbers, as those numbers remain comparable between 29 °C and control 25 °C. Notably, males at cold 18 °C and 29 °C had similarly increased frequencies of spermatid elongation and individualization defects which, considering the high sperm abundance and male fertility measured at 18 °C, indicate that spermatogenesis has a high tolerance for elongation and individualization defects. Interestingly, the abundance of sperm at 29 ° C decreases abruptly and with no evidence of apoptosis as they transition into the seminal vesicle near the end of spermatogenesis, pointing to sperm elimination through an unknown mechanism. Finally, sperm from males at 29 °C fertilize eggs less efficiently and do not support embryos past the first stage of embryogenesis, indicating that poor sperm quality is an additional cause of male infertility at 29 °C (O'Neill, 2023).

Evolutionary diversification reveals distinct somatic versus germline cytoskeletal functions of the Arp2 branched actin nucleator protein

Branched actin networks are critical in many cellular processes, including cell motility and division. Arp2, a protein within the 7-membered Arp2/3 complex, is responsible for generating branched actin. Given its essential roles, Arp2 evolves under stringent sequence conservation throughout eukaryotic evolution. This study unexpectedly discovered recurrent evolutionary diversification of Arp2 in Drosophila, yielding independently arising paralogs Arp2D in obscura species and Arp2D2 in montium species. Both paralogs are unusually testis-enriched in expression relative to Arp2. Whether their sequence divergence from canonical Arp2 led to functional specialization was investigated by replacing Arp2 in D. melanogaster with either Arp2D or Arp2D2. Despite their divergence, it was surprisingly found that both complement Arp2's essential function in the soma, suggesting they have preserved the ability to polymerize branched actin even in a non-native species. However, it was found that Arp2D -expressing males are subfertile and display many defects throughout sperm development. Two highly diverged structural regions in Arp2D were pinpointed that contribute to these defects: subdomain 2 and the C-terminus. It was expected that germline function would be rescued by replacing Arp2D's long and charged C-terminus with Arp2's short C-terminus, yet surprisingly, the essential somatic function of Arp2D was lost. Therefore, while Arp2D's structural divergence is incompatible with D. melanogaster sperm development, its unique C-terminus has evolved a critical role in actin polymerization. These findings suggest canonical Arp2's function differs between somatic versus germline contexts, and Arp2 paralogs have recurrently evolved and specialized for actin branching in the testis (Stromberg, 2023).

Antimony (Sb), is thought to induce testicular toxicity, although this remains controversial. This study investigated the effects of Sb exposure during spermatogenesis in the Drosophila testis and the underlying transcriptional regulatory mechanism at single-cell resolution. Firstly, it was found that flies exposed to Sb for 10 days led to dose-dependent reproductive toxicity during spermatogenesis. Protein expression and RNA levels were measured by immunofluorescence and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) was performed to characterize testicular cell composition and identify the transcriptional regulatory network after Sb exposure in Drosophila testes. scRNA-seq analysis revealed that Sb exposure influenced various testicular cell populations, especially in GSCs_to_Early_Spermatogonia and Spermatids clusters. Importantly, carbon metabolism was involved in GSCs/early spermatogonia maintenance and positively related with SCP-Containing Proteins, S-LAPs, and Mst84D signatures. Moreover, Seminal Fluid Proteins, Mst57D, and Serpin signatures were highly positively correlated with spermatid maturation. Pseudotime trajectory analysis revealed three novel states for the complexity of germ cell differentiation, and many novel genes (e.g., Dup98B) were found to be expressed in state-biased manners during spermatogenesis. Collectively, this study indicates that Sb exposure negatively impacts GSC maintenance and spermatid elongation, damaging spermatogenesis homeostasis via multiple signatures in Drosophila testes and therefore supporting Sb-mediated testicular toxicity (Yu, 2023).

Transcriptome analysis of several animal clades suggests that male reproductive tract gene expression evolves quickly. However, the factors influencing the abundance and distribution of within-species variation, the ultimate source of interspecific divergence, are poorly known. Drosophila melanogaster, an ancestrally African species that has recently spread throughout the world and colonized the Americas in the last roughly 100 years, exhibits phenotypic and genetic latitudinal clines on multiple continents, consistent with a role for spatially varying selection in shaping its biology. Nevertheless, geographic expression variation in the Americas is poorly described, as is its relationship to African expression variation. This study investigate these issues through analysis of two male reproductive tissue transcriptomes (testis and accessory gland) in samples from Maine (USA), Panama, and Zambia. Dramatic differences were found between these tissues in differential expression between Maine and Panama, with the accessory glands exhibiting abundant expression differentiation and the testis exhibiting very little. Latitudinal expression differentiation appears to be influenced by selection on Panama expression phenotypes. While the testis shows little latitudinal expression differentiation, it exhibits much greater differentiation than the accessory gland in Zambia vs. American population comparisons. Expression differentiation for both tissues is non-randomly distributed across the genome on a chromosome arm scale. Interspecific expression divergence between D. melanogaster and D. simulans is discordant with rates of differentiation between D. melanogaster populations. Strongly heterogeneous expression differentiation across tissues and timescales suggests a complex evolutionary process involving major temporal changes in the way selection influences expression evolution in these organs (Cridland, 2023).

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, mutations were engineered in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, it was found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl (see Drosophila Sxl), implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori (Yang, 2023).

Ageing is a complex biological process that is accompanied by changes in gene expression and mutational load. In many species, including humans, older fathers pass on more paternally derived de novo mutations; however, the cellular basis and cell types driving this pattern are still unclear. To explore the root causes of this phenomenon, this study performed single-cell RNA sequencing on testes from young and old male Drosophila and genomic sequencing (DNA sequencing) on somatic tissues from the same flies. Early germ cells from old and young flies were found to enter spermatogenesis with similar mutational loads but older flies are less able to remove mutations during spermatogenesis. Mutations in old cells may also increase during spermatogenesis. These data reveal that old and young flies have distinct mutational biases. Many classes of genes show increased postmeiotic expression in the germlines of older flies. Late spermatogenesis-biased genes have higher dN/dS (ratio of non-synonymous to synonymous substitutions) than early spermatogenesis-biased genes, supporting the hypothesis that late spermatogenesis is a source of evolutionary innovation. Surprisingly, genes biased in young germ cells show higher dN/dS than genes biased in old germ cells. These results provide new insights into the role of the germline in de novo mutation (Witt, 2023).

Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. This study describes a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. Similar premeiotic mitochondrial clusters were observed in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. It is suggested that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development (Li, 2023).

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. This study identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C(2) H(2) type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, it was found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92E^F06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, these results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila (Hu, 2023).

Centrosomes are multi-protein organelles that function as microtubule (MT) organizing centers (MTOCs), ensuring spindle formation and chromosome segregation during cell division. Centrosome structure includes core centrioles that recruit pericentriolar material (PCM) that anchors γ-tubulin to nucleate MTs. In Drosophila melanogaster, PCM organization depends on proper regulation of proteins like Spd-2, which dynamically localizes to centrosomes and is required for PCM, γ-tubulin, and MTOC activity in brain neuroblast (NB) mitosis and male spermatocyte (SC) meiosis. Some cells have distinct requirements for MTOC activity due to differences in characteristics like cell size or whether they are mitotic or meiotic. How centrosome proteins achieve cell-type-specific functional differences is poorly understood. Previous work identified alternative splicing and binding partners as contributors to cell-type-specific differences in centrosome function. Gene duplication, which can generate paralogs with specialized functions, is also implicated in centrosome gene evolution, including cell-type-specific centrosome genes. To gain insight into cell-type-specific differences in centrosome protein function and regulation, this study investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. This failure to accumulate and function in meiosis was mapped to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types (Rusan 2023).

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. This study shows that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. This study further demonstrates that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, these data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis (Iki, 2023).

Although the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. In this study, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans provides insight into molecular strategies that underlie hpRNA emergence and their potential roles in sex chromosome conflict. In particular, these data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, and recurrent targeting of testis HMG-box loci by hpRNAs. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since strong derepression of targets of the youngest hpRNAs was observed, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to speciation (Vedanayagam, 2023).

This study conceived two general classes of hpRNA targets. While hpRNAs are all evolutionary young, some of their targets are relatively old genes; e.g. targeting of ATP synthase-β by hpRNA-1. It is imagined that there are adaptive reasons for why mild suppression of such loci imparts beneficial regulatory consequences. This may be due directly to the acquisition of elevated transcriptional properties of the targets, and/or by emergence of duplicated loci with preferred testis expression, which appears to be a relatively common process. In any case, the role for endo-RNAi here is to modulate target activity, since full suppression of these genes is clearly deleterious. Still, it is speculated further that as several well-conserved targets of hpRNAs encode protein activities that have been linked to speciation, such as heterochromatin, DNA damage, and energy homeostasis, these conserved genes may harbor selfish activities in certain species that warrants adaptive suppression by RNAi. Since the miRNA pathway is not typically involved in adaptive targeting, and instead relies upon capture of targets bearing invariant miRNA seed matches, the RNAi pathway may be more flexible to suppress such genes. A counterpoint to this is the fact that specific testis-expressed, evolutionarily young, miRNA clusters diverge even within their seed regions. This suggests a possible atypical role for specialized miRNA loci in "hpRNA"-like evolutionary targeting dynamics (Vedanayagam, 2023).

Annotation of hpRNAs in two species indicates that, as a rule, these endogenous siRNA loci comprise very short-lived genes. Thus, they can at best only mediate modestly conserved regulation. If this is the case, can any general principles be learned from such fast-evolving regulatory networks? In fact, when considering this study alongside recent literature, this study found several recurrent themes that provide a framework for understanding how endogenous RNAi is harnessed in biology (Vedanayagam, 2023).

It was recently found that de novo hpRNAs in the simulans clade are required to silence a newly-emerged, amplifying, and selfish set of X-linked protamine derivatives, namely the Dox family. Protamines are central factors that condense the sperm genome, and therefore seem ripe for co-option by selfish factors to disrupt paternal inheritance. Indeed, following removal of histones, multiple sperm nuclear basic proteins (SNBPs) play roles in packaging sperm chromatin, and most of these contain HMG-box domains. Beyond the X-linked Dox family, there was found a separate amplification of X-linked tHMG box genes in D. simulans that are concomitantly associated with silencing by cognate hpRNAs. It is thus infererd that there is recurrent innovation of selfish SNBP activities by X chromosomes, consistent with the notion of sex chromosome meiotic drive that requires silencing by endogenous RNAi. Protamines are also functionally relevant to activity of Segregation Distorter, an autosomal meiotic drive system in D. melanogaster. Moreover, the Malik group recently reported high turnover of testis HMG-box loci across the Drosophilid genus, supporting the notion that their rapid evolution is due to recurrent intragenomic conflict between sex chromosomes. It is predictd that hpRNAs may be employed to silence other protamine-based meiotic drive phenomenon in other species (Vedanayagam, 2023).

Within the Dox superfamily system itself, this study documented the innovation of novel hpRNAs that bear chimeric domain structures characteristic of coding Dox genes, but that lack the HMG-box. Instead, they incorporate sequences from the piwi-interacting RNA (piRNA) factors Tapas and Krimper, also bear TE fragments. Homology to piRNA factors Tapas and Krimper were recognized in a prior study in distinct duplicate copies of the ancestral "ODox" gene, termed X:17.1 and X:17.2. However, the current analyses revealed that the duplicate copies of the ancestral ODox gene bear inverted repeats and now generate hpRNA-siRNAs (two independent loci, which are termed hp-ODox1 and hp-ODox2) in contemporary D. simulans. Overall, the genetic conflict that led to identification of Dox and Nmy is actually part of a far more extensive and rapidly evolving network of putative meiotic drivers and suppressors, and may in fact integrate activities of the siRNA and piRNA pathways. Relevant to this, the third recognized sex ratio meiotic drive system in D. simulans ("Paris"), is driven by HP1D2, a derivative of the core piRNA factor Rhino. Thus, there are recurrent linkages of piRNA factors to sex ratio meiotic drive, notwithstanding that TEs are themselves selfish genetic elements (Vedanayagam, 2023).

Of course, TEs are intrinsically selfish elements that are targeted by host genomic defenses, most famously by piRNAs. However, recent studies provide analogous conceptual involvement for co-option of the piRNA pathway by drive systems. As mentioned, the Paris SR system utilizes a de novo copy of the HP1-like factor Rhino, a central nuclear piRNA factor that defines piRNA cluster transcription. As another example, telomeric TART elements were found to have captured a fragment of Nxf2, a piRNA-specific copy of the mRNA export machinery that gained activity in co-transcriptional silencing. It is surmised that the capture of some piRNA factors by hpRNA loci may in fact reflect their selfish activities of such defense factors (Vedanayagam, 2023).

The adaptive deployment of hpRNAs in the testis in D. simulans highlights that some of the most important biologically overt manifestations of endo-RNAi cannot be studied in the major model system D. melanogaster. Looking to other Drosophilids, the recognition of rampant duplications of the RNAi effector AGO2 in various obscura clade species, resulting primarily in testis-restricted paralogs, provides a further hint into active genomic conflicts that may be playing out in these species. Indeed, intragenomic conflicts that mediate aberrant sex ratio and/or sterility, specifically in male fathers, have been documented in the obscura clade. The genetic factors in these conflicts remain to be documented fully, but it is intriguing to hypothesize whether de novo hpRNAs might be involved in any of these scenarios. Overall, the Drosophila RNAi/hpRNA pathway provides a policing system that helps to surveil and silence gene expression in the testis against selfish meiotic loci. It is speculated that such cycles of drive and repression are poised to underlie speciation (Vedanayagam, 2023).

The GAGA protein (also known as GAF) is a transcription factor encoded by the Trl gene in D. melanogaster. GAGA is involved in the regulation of transcription of many genes at all stages of fly development and life. Recently, the participation of GAGA in spermatogenesis was studied and it was discovered that Trl mutants experience massive degradation of germline cells in the testes. Trl underexpression induces autophagic death of spermatocytes, thereby leading to reduced testis size. This study aimed to determine the role of the transcription factor GAGA in the regulation of ectopic germline cell death. How Trl underexpression affects gene expression in the testes was examined. 15,993 genes were identified in three biological replicates of an RNA-seq analysis, and transcript levels were compared between hypomorphic Trl (R85)/Trl (362) and Oregon testes. A total of 2,437 differentially expressed genes were found, including 1,686 upregulated and 751 downregulated genes. At the transcriptional level, the development of cellular stress was detected in the Trl-mutant testes: downregulation of the genes normally expressed in the testes (indicating slowed or abrogated spermatocyte differentiation) and increased expression of metabolic and proteolysis-related genes, including stress response long noncoding RNAs. Nonetheless, in the Flybase Gene Ontology lists of genes related to cell death, autophagy, or stress, there was no enrichment with GAGA-binding sites. Furthermore, no specific GAGA-dependent cell death pathway was identified that could regulate spermatocyte death. Thus, these data suggest that GAGA deficiency in male germline cells leads to an imbalance of metabolic processes, impaired mitochondrial function, and cell death due to cellular stress (Fedorova, 2023).

Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. This study attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies (Kakino, 2023).

Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. This study presents data from the fruitfly Drosophila simulans that address these questions. A family of de novo, protamine-derived X-linked selfish genes (the Dox gene family: Distorter on the X-which is found on the X chromosome and kills Y chromosome-bearing sperm) was demonstrated to be silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. The Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, the first experimental evidence is provided that Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis (Vedanayagam, 2023).

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence was found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction (Matzkin, 2023).

Ejaculate proteins are key mediators of post-mating sexual selection and sexual conflict, as they can influence both male fertilization success and female reproductive physiology. However, the extent and sources of genetic variation and condition dependence of the ejaculate proteome are largely unknown. Such knowledge could reveal the targets and mechanisms of post-mating selection and inform about the relative costs and allocation of different ejaculate components, each with its own potential fitness consequences. This study used liquid chromatography coupled with tandem mass spectrometry to characterize the whole-ejaculate protein composition across 12 isogenic lines of Drosophila melanogaster that were reared on a high- or low-quality diet. New proteins were discovered in the transferred ejaculate, and their origin in the male reproductive system was inferred. It was further found that the ejaculate composition was mainly determined by genotype identity and genotype-specific responses to larval diet, with no clear overall diet effect. Nutrient restriction increased proteolytic protein activity and shifted the balance between reproductive function and RNA metabolism. These results open new avenues for exploring the intricate role of genotypes and their environment in shaping ejaculate composition, or for studying the functional dynamics and evolutionary potential of the ejaculate in its multivariate complexity (Zeender, 2023).

In species with internal fertilization, sperm, and seminal fluid are transferred from male to female during mating. While both sperm and seminal fluid contain various types of molecules, including RNA, the role of most of these molecules in the coordination of fertilization or in other possible functions is poorly understood. In Drosophila, exosomes from the accessory gland, which produces seminal fluid, are transferred to females, but their potential cargoes have not been described. Moreover, while the RNA composition of sperm has been described in several mammalian species, little work on this problem has occurred in Drosophila. This study used single nucleotide polymorphism differences between males and females from a set of highly inbred lines of D. melanogaster, and transcriptome data from the female reproductive tract, sperm, testis, and accessory gland, to investigate the potential origin, male vs female, RNA molecules isolated from 3 female reproductive tract organs, the seminal receptacle and spermatheca, which store sperm, and the parovaria, which does not. Mated females were found to carry male-derived transcripts from many genes, including those that are markers of the accessory gland and known seminal fluid proteins. These observations also support the idea that intact sperm transcripts can be isolated from the female sperm storage organs (Cridland, 2023).

In Drosophila and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). This study used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). Obp56g was found to be required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the male's ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. This study found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage. The evolutionary history of these seminal Obp genes was examined, as several studies have documented rapid evolution and turnover of SFP genes across taxa. Extensive lability in gene copy number and evidence were found of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time (Brown, 2023).

Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis, recent work showed that pro-niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which was found to be also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. The function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. This work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche (Anllo, 2022).

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. This study found that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. It is proposed that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation (Greenspan, 2022).

Whereas stem and progenitor cells proliferate to maintain tissue homeostasis, fully differentiated cells exit the cell cycle. How cell identity and cell-cycle state are coordinated during differentiation is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs). CySCs give rise to post-mitotic cyst cells, providing a tractable model to study the links between stem cell identity and proliferation. While cell-cycle progression was shown to be required for CySC self-renewal, the E2f1/Dp transcription factor is dispensable for self-renewal but instead must be silenced by the Drosophila retinoblastoma homolog, Rbf, to permit differentiation. Continued E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial biogenesis rescues the differentiation of CySCs with ectopic E2f1/Dp activity but not their cell-cycle exit. In sum, E2f1/Dp coordinates cell-cycle progression with stem cell identity by regulating the metabolic state of CySCs (Sainz de la Maza, 2022).

Loss of chic function in somatic cyst cells impaired the ability of cyst cells to build and/or maintain the cytoplasmic extensions through which they embrace and enclose spermatogonial cysts. Two somatic cyst cells normally surround each gonialblast and enclose its mitotic and meiotic progeny throughout Drosophila spermatogenesis. The cyst cells co-differentiate with the germ cells they enclose. Several lines of evidence support the model that either the ability of somatic cyst cells to enclose germ cells or their ability to send signals to adjacent germ cells is important to restrict proliferation and promote differentiation of germ cells. In either case, activation of EGFR in cyst cells is required for cyst cells to enclose germ cells and/or send the signals for germ cells to differentiate. The similarities in phenotype between loss of chic function and loss of EGFR activation in somatic cyst cells raise the possibility that chic/profilin may act downstream of activated EGFR to modulate the actin cytoskeleton for the remodeling of cyst cells to form or maintain the cytoplasmic extensions that enclose germ cells. Indeed, activated EGFR is known in other systems to tyrosine phosphorylate phospholipase C-γ1 (PLC-γ1), a soluble enzyme in quiescent cells like daughter cyst cells, activating it to catalyze hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), which binds profilin protein with high affinity, which inhibits the interaction between profilin and actin. The hydrolysis of PIP₂ by activated PLC-γ1 results in localized release of profilin and other actin-binding proteins, enabling them to interact with actin and participate in cytoskeletal rearrangement and membrane protrusion. Thus, based on biochemical analysis in other systems, a link between EGFR activation and profilin leading to local remodeling of the actin cytoskeleton is plausible in somatic cyst cells, although it remains to be directly tested (Shields, 2014).

These data strongly indicate that the secondary F-actin ring must be disassembled for abscission to be reinitiated. This suggests that F-actin at the IC bridge inhibits abscission, and work in other cells supports this. Inhibition of the Cofilin phosphatase, activation of AurB, depletion of phosphoinositide 5-phosphatase, or of Rab35 all lead to retention of F-actin at the IC bridge and inhibit abscission. Importantly, abscission could be restored after Rab35 depletion by forcing F-actin disassembly (Lenhart, 2015).

GSC-Gb pairs depleted for aurB fail to complete abscission prior to mitotic entry and form interconnected germ cells attached to the hub. This could suggest that AurB is normally required to promote abscission. However, expressing an activated form of Svn did not induce precocious abscission as would be expected in this model. Rather, Svn^S125E expression advanced the transition from phase one to two, while aurB depletion delayed it. These reciprocal effects suggest instead that AurB times the phase one-phase two transition. In this model, the lack of abscission in aurB mutants is an indirect consequence of spending a shorter fraction of the total cycle in phase two. For example, this study has shown that ESCRTIII is localized during phase two and in the apparent absence of central spindle microtubules. In aurB-depleted cells, there simply may not be enough time during the shortened phase two for the already compromised recruitment of ESCRTIII machinery to promote abscission prior to mitotic entry. It is also noted that the lack of a central spindle raises the issue of how ESCRTIII components are delivered to the IC bridge. Perhaps the midbody performs this role, as has been suggested for the C. elegans first cell division (Lenhart, 2015).

Recent studies have found that shrub is negatively regulated by AurB in female GSCs (Matias, 2015). Although the current results suggest that AurB activity should promote ESCRTIII function in the testis, it is compelling to speculate that AurB might control the phase one-phase two transition through shrub. Alternatively, AurB could directly control this transition by regulating disassembly of the secondary F-actin ring, as there is precedent for AurB controlling actin dynamics. For example, in the 'No Cut' pathway, maintenance of AurB activity late in cytokinesis is associated with persistence of F-actin at the IC bridge. Intriguingly, AurB can phosphorylate formin proteins and thereby regulate actin stress fiber formation. Although in this context AurB activity positively regulates actin polymerization, the interaction between AurB and formin suggests a direct link between CPC activity and actin dynamics. This connection is particularly compelling given that formins can also promote severing of actin filaments. Thus, it is intriguing to speculate that AurB phosphorylation of formins at the IC bridge in GSC-Gb pairs may promote severing of actin filaments in the secondary ring and thereby promote transition from phase one to phase two of delay (Lenhart, 2015).

Perhaps most excitingly, this study has identified non-autonomous control over GSC-Gb abscission by somatic cell encystment. This sheds light on the functional relevance of abscission delay. Encystment of spermatogonia by two somatic cells is required for proper germ cell differentiation. However, GSCs and their flanking CySCs do not coordinate daughter cell production by synchronizing their cell cycles. Linking abscission to encystment is an elegant alternative for promoting coordinated release of stem cell daughters from the niche (Lenhart, 2015).

Several questions are raised by the current observations, such as precisely when abscission is triggered relative to cyst cell engulfment of the Gb. It would be necessary to carry out live imaging simultaneously on germline and adjacent somatic cells to address this. However, imaging CySCs and cyst cells is fraught with difficulty due to their irregular morphology and small size. Thus, it has not yet been possible to image somatic cells with anywhere near the resolution achieved for GSC-Gb pairs (Lenhart, 2015).

Encystment could promote abscission through contact-dependent signaling, where CySCs or cyst cells produce the ligand. Alternatively, the abscission trigger might be mechanical, because tension has been suggested to regulate abscission in cultured cells. Here, as daughter cells migrated apart in culture following mitosis, tension along the bridge connecting them increased and this lengthened the time to abscission. Experimentally decreasing bridge tension triggered earlier abscission. In the current system, most Gbs are displaced some distance from the hub during phase two, with a consequent elongation of the IC bridge connecting those cells to the GSC. Perhaps movement of the Gb away from its mother GSC generates increased tension along the bridge. Symmetric encystment might relieve that tension by providing equalizing forces on both sides of the IC bridge, inducing abscission while ensuring that the Gb is properly associated with two somatic cells. In culture, increased tension delayed abscission by disrupting assembly of functional ESCRTIII complexes at the IC bridge. Therefore, it will be interesting to address whether ESCRTIII complexes in GSCs are temporally regulated by encystment. Whatever the mechanism, the cyst cells are clearly poised for intimate contact at the appropriate time, because the midbody remnant is sometimes taken up byÊencysting somatic cells after abscission (Lenhart, 2015).

This work has clarified the mechanism by which cytokinesis is delayed in GSCs, identifying three distinct regulatory events layered on top of the traditional program of cytokinesis. These events impose an appropriate delay, a timed reinitiation, and a regulated abscission in the GSCs. This stem cell-specific program assists in the coordinate release of differentiating daughter cells from the resident stem cell populations in this niche. Because similar requirements for synchronized daughter cell production between multiple stem cell populations exist in other tissues, it is enticing to speculate that regulated abscission might be used to promote coordination in other niches. Membrane scission is difficult to demonstrate in vivo in many systems, so it is not yet known if stem cells other than the germline exhibit abscission delay. As higher resolution methods are developed to visualize stem cell dynamics within endogenous niches, it will be interesting to see if abscission delay emerges as a conserved mechanism of niche-dependent control over stem cell proliferation (Lenhart, 2015).

The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. This study focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. A FACS-based approach was employed for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. Increased expression was found of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. This comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility (Adashev, 2022).

Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) 'eat-me' signals on target cells initiate the process, the precise sequence of events is largely unknown. This study shows that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, it was demonstrated that cyst cell-derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors (Zohar-Fux, 2022).

Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, this study showed that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo(-/-) GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo^-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. These results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution (Tseng, 2022).

This work reveals an unexpected model of GSC competition that results in biased inheritance. These results provide a mechanistic demonstration of the postulated 'mitotic drive' by which GSCs with a competition advantage transmit competitive alleles at a greater than the expected 50% Mendelian ratio. Studies in plants, yeast, flies, and mice have shown that selfish genetic elements can cause gene drive through various molecular mechanisms. These include 'meiotic drivers' that co-opt meiotic divisions or that kill viable gametes that do not inherit the selfish element. 'Mitotic drive' occurs earlier in the germline lineage, at the stem cell level, and is an understudied area. Prior work in the Drosophila ovary has shown that dedifferentiation-defective female GSCs 'win' by upregulating E-Cad at the GSC-niche interface and gradually pushing WT GSCs out of the niche. Since differentiation-defective GSCs do not differentiate into gametes, this study could not examine biased allele inheritance. Female GSCs with 4x gene dose of Myc were reported to be 'winners,' but biased inheritance was not assessed (Tseng, 2022).

Given the competitive advantage of GSCs lacking chinmo, why has evolution not selected for male GSCs with no chinmo expression? Since chinmo is an essential gene required for development (Zhu et al., 2006), loss of chinmo in GSCs might cause reduced chinmo expression in other tissues, likely reducing organismal fitness. Furthermore, chinmo^- dependent competition is a progressive phenotype requiring at least 2 weeks of adulthood. If males with mosaic chinmo^- clones in the germline were mated as young adults, both the chinmo⁺ and chinmo^- allele would be passed on to offspring, and this would be sufficient to maintain the chinmo⁺ allele in the population. Although the GSC pool in testes with chinmo^- cells is often monoclonal, why is the chinmo^- allele is not passed on to 100% of offspring? Males are maintained as virgins until their testes are dissected or until mating. This means that both chinmo^- GFP-negative and ubi-GFP-positive spermatids are stored in the seminal vesicle throughout the male's lifetime, and the single round of mating in these experiments allows for the transmission of both types of sperm (Tseng, 2022).

This work identified three phenotypes-competition, aging, and transdifferentiation-that are dependent on ectopic Pcan expression in chinmo^-deficient stem cells. This remarkable finding raises the important question of which factors regulate Chinmo expression in GSCs during adulthood and which genes are direct targets of Chinmo. This study identified chinmo as a JAK/STAT target gene, but STAT-deficient GSCs still express Chinmo protein, indicating that as-yet unidentified factors regulate Chinmo in GSCs and perhaps in other stem cells. Additionally, future molecular work will be needed to determine whether Pcan, Dg, and βPS are direct Chinmo target genes in germline and somatic stem cells (Tseng, 2022).

This work raises the possibility that other mutant stem cells can 'cheat' by resculpting their microenvironment and then ensuring their own retention in this remodeled milieu. Paternal age effect disorders (PAEs) encompass a broad spectrum of spontaneous congenital disorders and are thought to arise from rare selfish GSCs that are positively selected and clonally expanded. While the current model of PAE postulates increased proliferation of mutant GSCs as the competitive mechanism, other selfish cellular behaviors, such as the ones this study has discovered, could also be functioning in the mammalian testis. Cancer stem cells could utilize the mechanisms described in this study to colonize a tissue. Leukemic stem cells induce progressive remodeling of the bone marrow niche, and this altered niche favors the mutant stem cells while impairing normal hematopoietic stem cell residence and contributes to bone marrow fibrosis. In sum, this work raises the possibility that selfish stem cells across species cheat using the mechanisms this study has discovered in competitive GSCs in the Drosophila testis (Tseng, 2022).

Although this study found significant increases in Pcan, Dg, and βPS transcripts in GSCs deficient for Chinmo, HCR-FISH analyses of relative intensity are not strictly quantitative. As such, it cannot be ruled out that Chinmo affects other aspects of mRNA regulation, such as splicing. Because the transcriptome of chinmo^-/- GSCs have not been attained at sufficient resolution and this study has not been able to perform Chinmo ChIP-seq in GSCs, it is not possible to conclude that Chinmo directly represses these genes. It will be important in the future to determine Chinmo occupancy on chromatin in GSCs and in other stem cells. Surprisingly, the niche spaces vacated by non-mutant neighbor GSCs are occupied by cyst stem cells (CySCs) and not by chinmo^-/- GSCs. It is not understood why the CySCs predominate, and future studies employing ex vivo live-imaging will be important to gain insights into this process (Tseng, 2022).

The regulation of the cell division cycle is governed by a complex network of factors that together ensure that growing or proliferating cells maintain a stable genome. Defects in this system can lead to genomic instability that can affect tissue homeostasis and thus compromise human health. Variations in ploidy and cell heterogeneity are observed frequently in human cancers. This study examined the consequences of upregulating the cell cycle regulator Cyclin E in the Drosophila melanogaster male accessory gland. The accessory gland is the functional analog of the human prostate. This organ is composed of a postmitotic epithelium that is emerging as a powerful in vivo system for modelling different aspects of tumor initiation and progression. Cyclin E upregulation in this model was shown to be sufficient to drive tissue dysplasia. Cyclin E overexpression drives endoreplication and affects DNA integrity, which results in heterogeneous nuclear and cellular composition and variable degrees of DNA damage. Evidence is presented showing that, despite the presence of genotoxic stress, those cells are resistant to apoptosis and thus defective cells are not eliminated from the tissue. It was also shown that Cyclin E-expressing cells in the accessory gland display mitochondrial DNA aggregates that colocalize with Cyclin E protein. Together, these findings show that Cyclin E upregulation in postmitotic cells of the accessory gland organ causes cellular defects such as genomic instability and mitochondrial defects, eventually leading to tissue dysplasia. This study highlights novel mechanisms by which Cyclin E might contribute to disease initiation and progression (Molano-Fernandez, 2022).

Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. The COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. By establishing colcemid-induced microtubule depolymerization as a sensitive assay, this study examined the characteristics of COC activity and found that it functions uniquely in GSCs but not in their differentiating progeny. The COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types (Venkei, 2015).

Stereotypical orientation of the mitotic spindle is a widely utilized mechanism to achieve asymmetric cell division. Although considerable knowledge has accumulated regarding how spindle orientation is established, little is known about whether cells possess a mechanism that monitors successful spindle orientation. In the present study, using colcemid treatment as a sensitive assay, the nature of the COC, which is the only known orientation/polarity checkpoint in multicellular organisms, was was investigated. It was established that: (1) the COC specifically operates in GSCs, but not differentiating germ cells (GBs and SGs); (2) the COC operates in G2 phase of the cell cycle to prevent precocious entry into mitosis upon centrosome misorientation; and (3) as a checkpoint mechanism, the COC is distinct from the SAC (Venkei, 2015).

These results show that the COC is a GSC-specific checkpoint that monitors centrosome orientation and arrests cells in G2 phase when centrosomes are not correctly oriented. It remains unclear whether the COC-mediated G2 arrest might eventually undergo adaptation to allow mitotic entry, as is the case with mitotic slippage in the SAC. It is worth noting in this context that the GSC mitotic index never increased during 6 h of colcemid treatment, whereas SGs seem to undergo mitotic slippage by 6 h of colcemid treatment. This suggests that the COC-mediated G2 arrest is relatively strong. The findings that the mad2 mutation has no effect on G2 arrest of GSCs and that par-1 and cnn mutants have no effect on mitotic arrest in GBs/SGs upon colcemid treatment strongly support the notion that the COC and SAC constitute distinct checkpoint mechanisms. Although CySCs and SGs also orient their mitotic spindles, the present study shows that spindle orientation in these cells is not under the control of the COC. However, the lack of a COC in these cell types does not exclude the possibility that distinct polarity checkpoint mechanisms are in place to ensure correct spindle orientation. If the arrest points of such checkpoints are not prior to the arrest point of the SAC (i.e. metaphase), the assay using colcemid would not reveal their presence (Venkei, 2015).

Mutant analysis of multiple centrosomal components in this study revealed a selective requirement for centrosomal components in the function of the COC. Sas-4 and Cnn are crucial for COC function, whereas Spd-2 and Apc1 are not. This indicates that not all of the centrosomal proteins are involved in COC function. Conversely, not all COC components are localized to the centrosome. As shown in a previous study, an essential component of the COC, Par-1, is localized to the spectrosome, where it regulates the localization of Cyclin A to regulate mitotic entry. How the information on centrosomal orientation is communicated to the spectrosome, where Par-1 and Cyclin A localize, remains to be determined. A major outstanding question in understanding the COC is how it senses the location of the centrosome with respect to the hub cells. Previous studies have shown that the mother centrosome is anchored to the adherens junctions formed at the hub-GSC interface via MTs. Therefore, it is plausible that the COC senses aspect(s) of these interactions. It awaits future investigation to understand how the association of the centrosome with the hub-GSC interface is mechanistically sensed, and how such information is integrated with the activity of COC component(s) on the spectrosome (Venkei, 2015).

In summary, this present work establishes that the COC is a checkpoint mechanism that is distinct from the SAC and monitors correct centrosome orientation specifically in GSCs. It is speculated that a similar mechanism might be in place in other systems that rely on asymmetric cell division (Venkei, 2015).

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. This study identified PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). It is proposed that PHF7 acts as a conserved epigenetic 'reader' that activates the male germline sexual program (Yang, 2012).

Sex determination is key to sexual reproduction, and both somatic cells and germ cells need to establish sex-specific developmental fates. Germline sexual development is essential for the production of two distinct gametes, and underlies important differences in the regulation of male versus female fertility. In some species, germline stem cells are present in both males and females to sustain constant gamete production, but are regulated differently throughout development. In other species such as humans, sex-specific germ cell development produces a germline stem cell population only in males, whereas females have a much more limited capacity in making eggs. Defects in germline sexual development lead to a failure in gametogenesis, thus the study of germline sex determination is essential for understanding normal reproductive potential and treating infertility (Yang, 2012).

In some animals, such as mammals and Drosophila, the sex chromosome compositions of the soma and germline are interpreted independently, and the 'sex' of the germline must match that of the soma for proper germ cell development to occur. For example, patients with Klinefelter's Syndrome have an XXY sex chromosome constitution and are almost always infertile. These individuals develop somatically as males due to the presence of a Y chromosome but the germline suffers from severe atrophy, including the loss of premeiotic germline and germline stem cells. This is due to the presence of two X chromosomes in the germ cells, as the limited spermatogenesis in these patients is from germ cells that have lost one of the X chromosomes. In Drosophila, XX females that are somatically transformed into males exhibit a similar germline loss due to a conflict in sexual identity between the masculinized soma and XX germline. Thus, fruit flies are a valuable model organism for studying how germ cells establish a proper sexual identity by coordinating intrinsic signals and those coming from the soma (Yang, 2012).

In Drosophila, the presence of two X chromosomes promotes female somatic identity by activating an alternative splicing cascade that acts through Sex lethal (SXL) and Transformer (TRA), and ultimately leads to production of either the male or female forms of the transcription factors Doublesex (DSX) and Fruitless (FRU). DSX and FRU are responsible for virtually all sexually dimorphic somatic traits in Drosophila, with DSX being the key factor in the somatic gonad. In contrast, the germline does not determine its sex with this cascade and factors like TRA and DSX are not required in germ cells. Although SXL is required to promote female germ cell identity, its targets and mechanism of action in the germline are not known. The transcription factor OVO and the ubiquitin protease Ovarian Tumor (OTU) are also required in the female germline and thought to function upstream of SXL. Even less is known about how sexual identity is specified in male germ cells. Male germ cells receive a signal through the JAK/STAT pathway that promotes their sexual identity, but the downstream factors that are subsequently activated are not known. Similarly, how male germ cells respond to their own sex chromosome constitution is also not known (Yang, 2012).

This study reports a histone code reader, Plant Homeodomain (PHD) Finger 7 (PHF7), that acts in the Drosophila germline to promote male sexual identity. PHF7 is specifically expressed in male germ cells from early stages of development and is restricted to male germline stem cells (GSCs) and spermatogonia. Phf7 is required for GSC maintenance and proper entry into spermatogenesis. Interestingly, expression of Phf7 in female germ cells causes ablation of the female germline. Moreover, Phf7 affects sexual compatibility between germline and soma. Loss of Phf7 in XY germ cells alleviates the germline loss typically observed when XY germ cells are surrounded by a female soma, and expression of Phf7 can induce spermatogenesis in XX germ cells nurtured by male soma. These findings indicate that Phf7 is an essential factor in determining sexual development in the Drosophila germline, and suggest that activation of the male identity occurs through interaction with the germline epigenome (Yang, 2012).

The data indicate that Phf7 acts to promote a male identity in the germline. Loss of Phf7 function affected male GSC maintenance and spermatogenesis, but had no effect in females. Phf7-mutant GSCs exhibited a more female-like pattern of spectrosome localization, and male (XY) germ cells mutant for Phf7 were more compatible with a female soma than were wild-type male germ cells. Further, expression of PHF7 was able to masculinize the female germline: PHF7 expression induced apoptosis in developing XX germ cells and interacted with mutations in otu in a manner that indicates XX germ cells that express PHF7 are more male-like. Strikingly, PHF7 expression was able to induce spermatogenesis in XX germ cells when they are present in a male soma, something that XX germ cells are normally not able to do. Taken together, these results indicate that Phf7 promotes and is sufficient to induce male identity in the germline (Yang, 2012).

Sex determination is thought to be initiated early during development, and sex-specific differences in the male and female germline are first observed during embryogenesis. The data indicate that Phf7 plays a role in early germline sexual development, rather than a late role to regulate germ cell differentiation and gametogenesis. First, PHF7 expression is observed in the embryonic gonad and, in the adult, PHF7 is found in the GSCs and early gonia and disappears dramatically as gonia transition to spermatocytes. Further, forced PHF7 expression disrupts early female germ cell development, around the time when they are first forming GSCs. Expression of PHF7 after the early cystoblast stage (Bam-Gal4, UAS-Gal4) had no effect on the female germline, indicating that it can only affect early stages of female germ cell development. Phf7 mutants show defects in male GSC behavior and maintenance, and in the initial progression to form spermatocytes, but it is possible that these defects are due to even earlier problems in male sexual identity (Yang, 2012).

Germline sexual identity is determined by both the germ cell sex chromosome constitution and signals from the surrounding soma. Phf7 expression is activated in XX germ cells when in contact with a male soma and repressed in XY germ cells when contacting a female soma. However, in a female somatic environment, XY germ cells are somewhat more likely than XX germ cells to express Phf7, indicating that Phf7 may also respond to the sex chromosome constitution of the germ cells in addition to being regulated by the soma. Further, exogenous expression of Phf7 is required to promote spermatogenesis in XX germ cells when in a male soma. Thus, the Phf7 expression that is normally initiated in such germ cells by the male soma must either not be maintained, or may be insufficient to overcome the influence of the XX sex chromosome genotype (Yang, 2012).

It is likely that Phf7 is not acting alone to control male sexual identity. Phf7 mutant males are still able to undergo spermatogenesis, but at a much reduced capacity. This appears to be the null phenotype for Phf7 as ther mutants have lost significant portions of the coding sequence. Further, when PHF7 is expressed in XX germ cells present in a male soma, these germ cells can undergo spermatogenesis, but the penetrance of this phenotype is low. Interestingly, the rescue of spermatogenesis in these XX germ cells follows an 'all or nothing' pattern; either the rescue is largely complete to give full testes and sperm production, or little rescue is observed. Therefore, there appears to be a threshold that must be crossed to promote male germline sexual identity, and that once this threshold is met, those germ cells either take over the testis, or induce other germ cells to also follow the male pathway. The simplest explanation for both the incomplete block to spermatogenesis in Phf7 mutants and the incomplete rescue of spermatogenesis by Phf7 in XX males is that an additional factor (or factors) exists that promotes male identity in addition to Phf7. Such a factor could function parallel to Phf7 in a single pathway, or represent independent input regarding germline sex determination (e.g., independent signals from the soma that influence germline sex) (Yang, 2012).

PHD fingers, such as those found in PHF7, are best known for their ability to specifically bind histones that have been modified on their N-terminal tails, in particular methylated H3K4. This study shows that both Drosophila and human PHF7 can directly associate with dimethylated H3K4, indicating that PHF7 is indeed a histone code reader. It is uncommon for PHD domains to associate preferentially with H3K4me2 over H3K4me3, but this specificity has been observed previously, and is likely important for how PHF7 modulates expression of its targets. Both di- and trimethylated H3K4 are found at actively transcribed genes, but H3K4me2 is normally localized at the 5′ end of coding sequences, downstream of H3K4me3, which is near promoters. The two marks are also regulated by different demethylases. A few recent studies have started to dissect effects of H3K4me2 on gene transcription, but the exact mechanisms are not well understood. Some PHD finger proteins also contain other domains, such as those that modify histones enzymatically. This does not appear to be the case for PHF7, and the region of homology between PHF7 homologs of different species is restricted to the PHD domains. However, individual PHD fingers can bind modified histone tails independently, and it is yet unclear which PHD finger in PHF7 contacts H3K4me2 and what activities the others might have. The logic of how PHF7 is recruited to specific loci and affects chromatin structure and gene activity are interesting questions for future work (Yang, 2012).

Another point of interest is how a reader of such a common epigenetic mark would have a sex-specific role in regulating male germline identity. It has been observed that mutation of an H3K4me2 demethylase in Caenorhabditis elegans, which leads to increased dimethylation at H3K4, results in ectopic activation of male-specific germline genes. A similar mutation in Drosophila causes female germline developmental defects, which may be related to the germline atrophy observed when PHF7 expression was upregulated in female germ cells. These data are consistent with the hypothesis that H3K4me2 has a role in regulating the male germline genome. Interestingly, another germline chromatin factor, No child left behind (NCLB), has been identifed that is expressed in germ cells of both sexes but required for GSC function only in males. Thus, NCLB may cooperate with PHF7 in regulating the male GSC transcriptional program (Yang, 2012).

Based on sequence homology, orthologs of Phf7 are present in a wide range of mammalian species. Human and mouse PHF7 share extensive homology to Drosophila PHF7 throughout the N-terminus where the PHD fingers are present, and the results confirm that human PHF7 recognizes H3K4me2, similar to the fly protein. Interestingly, EST profiling indicates strong testis biases for Phf7 expression in many species, including humans, mice, rats, and dogs. Moreover, several studies that performed genome-wide RNA profiling from purified mouse germline populations indicate that mouse Phf7 expression is present in spermatogonia and is further induced in spermatocytes. Remarkably, human PHF7 was able to rescue fecundity defects in male flies mutant for Phf7. Thus, the sequence conservation observed between mammalian and Drosophila Phf7 represents true functional orthology (Yang, 2012).

As in Drosophila, germline sex determination in mouse is regulated at an early stage and is controlled by important signals from the soma, which for the mouse include retinoic acid and FGF9. Such signals regulate the timing of meiotic entry, which is different between the sexes, and also influence sex-specific programs of germline gene expression, such as expression of the key male-specific factor nanos2. Significant changes in germ cell chromatin occur during this critical time in germ cell development, including changes in the H3K4 methylation state. Thus, Phf7 represents a prime candidate for interpreting these chromatin changes in a sex-specific manner to regulate male-specific gene expression. It will be of great interest to determine whether Phf7 plays a critical role in mouse and human male germ cell development, as is proposed for Drosophila (Yang, 2012).

Doublesex (Dsx) and Fruitless (Fru) are the two downstream transcription factors that actuate Drosophila sex determination. While Dsx assists Fru to regulate sex-specific behavior, whether Fru collaborates with Dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs. This study reports that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously-studied aspects of sex-specific Fru expression, which is regulated by Transformer (Tra)-mediated alternative splicing, this study shows that male-specific expression of fru in the gonad is regulated downstream of dsx, and is independent of tra. fru genetically interacts with dsx to support maintenance of the niche throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, this study identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the niche, indicating that fru is likely a direct target for transcriptional regulation by Dsx. These findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra (Zhou, 2021).

In sexually reproducing animals, the proper production of gametes and successful copulation are equally critical for reproductive success. It is therefore important that both the gonad and the brain know their sexual identity. The Doublesex/Mab-3 Related Transcription Factors act downstream of sex determination and play an evolutionarily conserved role to establish and maintain sexual dimorphism in the gonad. Meanwhile, sexual dimorphism in other tissues such as the brain is controlled, to varying degrees in different animals, through autonomous control by the sex determination and non-autonomous signaling from the gonads. In many invertebrate species, another sex-determination gene fruitless (fru), which encodes multiple BTB-Zinc finger transcription factors, plays a central role in controlling mate choice, courtship behavior and aggression. How sex determination in the gonad and the nervous system are related and coordinated in these species remains unclear (Zhou, 2021).

The founding member of the DMRT family is Drosophila doublesex (dsx). dsx and fru undergo sex-specific alternative mRNA splicing by the sex determination factor Transformer (Tra), together with its co-factor Transformer-2 (Tra-2), to produce transcripts encoding sex-specific protein isoforms. It was once thought that dsx controls sexual dimorphism outside the nervous system while fru regulates sex-specific nervous system development and behavior. But more recent evidence shows that dsx cooperates with fru to specify sex-specific neural circuitry and regulate courtship behaviors. However, whether fru acts along with dsx to control sexual dimorphism outside the nervous system remains unknown (Zhou, 2021).

The fru gene locus contains a complex transcription unit with multiple promoters and alternative splice forms (see Fruitless is expressed male-specifically in the germline stem cell niche and is independent of Fru^M). Sex-specific regulation of fru was only known to occur through alternative splicing of transcripts produced from the P1 promoter, which produces the FruM isoforms. The downstream promoters (P2-P4) produce Fru isoforms (collectively named FruCom) encoded by transcripts that are common to both sexes and are required for viability in both males and females. fru P1 transcripts have only been detected in the nervous system, suggesting that sex-specific functions of fru are limited to neural tissue. However, FruCom is expressed in several non-neural tissues, including sex-specific cell types of the reproductive system. Further, from a recent genome-wide search for putative Dsx targets, fru was identified as a candidate for transcriptional regulation by Dsx. These data raise the possibility that fru functions cooperatively with dsx to regulate gonad development (Zhou, 2021).

Over the past decades, much effort has been focused on understanding the functions of fru in regulating sex-specific behaviors, yet it remained unclear whether fru plays a role in regulating sexual dimorphism outside the nervous system. The work presented in this study demonstrates that Fru is expressed male-specifically in the gonad stem cell niche, and is required for CySC maintenance, cyst cell survival, and for the maintenance of the hub during larval development. Further, male-specific expression of Fru in the gonad is independent of the previously described mechanism of sex-specific alternative splicing by Tra, and is instead dependent on dsx. fru appears to be a direct target for transcriptional regulation by Dsx. This work provides evidence that fru regulates sex-specific development outside the nervous system and alters traditional thinking about the structure of the Drosophila sex determination pathway (Zhou, 2021).

While it was previously reported that fru is expressed in tissues other than the nervous system, including in the gonad, a function for fru outside the nervous system was previously unknown. This study found that Fru is expressed in the developing and adult testis in the hub, the CySC, and the early developing cyst cells. Importantly, it was found that fru is important for the proper function of these cells (Zhou, 2021).

Fru is not expressed at the time of hub formation during embryogenesis, but expression is initiated during the L2/L3 larval stage. This correlates with a time period when the hub must be maintained and resist transforming into female niche structures: in dsx mutants, all gonads in XX and XY animals develop hubs, but in half of each, hubs transform into terminal filament cells and cap cells. fru is not required for initial hub formation, consistent with it not being expressed at that time. fru is also not, by itself, required for hub maintenance under the conditions that were possible to assay (prior to the pupal lethality of fru null mutant animals). However, under conditions where hub maintenance is compromised by loss of dsx function, fru clearly plays a role in influencing whether a gonad will retain a hub, or transform into TF. Fru expression in dsx mutant gonads correlates with whether they formed male or female niche structures, and removing even a single allele of fru is sufficient to induce more hubs to transform into TFs. Finally, ectopic expression of Fru in females is sufficient to inhibit TF formation and partially masculinize the gonad, but does not induce hub formation. Thus, it is proposed that fru is one factor acting downstream of dsx in the maintenance of the male gonad stem cell niche, but that it acts in combination with other factors that also regulate this process (Zhou, 2021).

This study also demonstrated that fru is required for CySC maintenance and for the survival of differentiating cyst cells. Loss of fru from the CySC lineage led to rapid loss of these CySCs from the testis niche. Since precocious differentiation of CySCs or an increase in their apoptosis was not observed, these mechanisms do not appear to contribute to CySC loss. One possibility is that fru is needed for CySCs to have normal expression of adhesion proteins and compete with other stem cells for niche occupancy. It has been shown that fru regulates the Slit-robo pathway and that robo1 is a direct target of fru in the CNS. Interestingly, the Slit-Robo pathway also functions in the CySCs to modulate E-cadherin levels and control the ability of CySCs to compete for occupancy in the niche. Therefore, fru may use similar mechanisms to maintain CySC attachment to the hub. fru also influences survival in the differentiating cyst cells, as an increase in cell death was observed in these cells in fru mutants. Several reports have demonstrated that fru represses programmed cell death in the nervous system. It was further indicated that the cell death gene reaper is a putative target of Fru. Thus, fru may play a role in repressing the apoptosis of cyst cells (Zhou, 2021).

In summary, fru function is clearly important for male niche maintenance and the function of the CySCs and their differentiating progeny. This provides clear evidence that fru regulates sex-specific development in tissues other than the nervous system. Whether additional tissues are also regulated by fru remains to be determined (Zhou, 2021).

Previously, it was thought that the only mechanism by which sex-specific functions of fru were regulated was through Tra-dependent alternative splicing of the P1 transcripts. fru null alleles are lethal in both sexes and Fru proteins derived from non-P1 promoters were thought to be sex-nonspecific and not to contribute to sex determination. Thus, fru and dsx were considered as parallel branches of the sex determination pathway, each independently regulated by Tra. This study demonstrates that fru can also be regulated in a manner independent of tra and dependent on dsx, and provides evidence that fru is a direct target for transcriptional regulation by Dsx (see Proposed model of the Drosophila sex determination pathway. First, Fru expression in the testis is independent of the P1 transcript that is regulated by Tra. A P1 Gal4 reporter is not expressed in the testis and a mutation that prevents FruM expression from P1 does not affect Fru immunoreactivity in the testis. Second, in animals that simultaneously express the female form of tra (Tra on) and the male form of Dsx [XX; dsxD/Df(3R)dsx3], Fru is expressed in the male mode in the testis, demonstrating that it is regulated by dsx and not tra. Finally, an evolutionarily conserved Dsx consensus binding site upstream of the P4 promoter is required for proper expression levels of a fru P4 reporter in the testis. Together, these data demonstrate a novel mode for fru regulation by the sex determination pathway, where sex-specific expression of fru is regulated by dsx. It also means that the large number of fru transcripts that do not arise from the P1 promoter can be expressed in a sex-specific manner to contribute to sexual dimorphism (Zhou, 2021).

The male and female forms of Dsx contain the same DNA binding domain and can regulate the same target genes, but often have opposite effects on gene expression. Prior to this study, the documented Dsx targets (Yolk proteins 1 and 2, bric-a-brac and desatF), along with other proposed targets, were all expressed at higher levels in females than males. Thus, for these targets, DsxF acts as an activator and DsxM acts as a repressor (or DsxM has no role). Interestingly, fru is the first identified Dsx target that is expressed in a male-biased manner. Thus, for direct regulation of fru, DsxM would activate expression while DsxF represses. Mechanistically for Dsx, this implies that the male and female isoforms are not dedicated repressors and activators, respectively, but may be able to switch their mode of regulation in a tissue-specific or target-specific manner. Mouse DMRT1 has also been shown to regulate gene expression both as transcriptional activator and repressor. Thus, it is quite possible that bifunctional transcriptional regulation is a conserved characteristic of DMRTs (Zhou, 2021).

It is possible that dsx regulation of fru occurs in the nervous system as well, where it co-exists with direct regulation of fru alternative splicing by Tra. It was originally thought that alternative splicing of the fru P1 transcript by tra was essential for male courtship behavior. However, more recently it was found that these animals could exhibit male courtship behavior if they were simply stimulated by other flies prior to testing. Interestingly, the courtship behavior exhibited by these males was dependent on dsx. It is proposed that fru might still be essential for male courtship in these fru P1-mutants, but that sex-specific fru expression is dependent on transcriptional regulation of other fru promoters by Dsx (Zhou, 2021).

If sex-specific fru function can be regulated both through alternative splicing by Tra and through transcriptional regulation by Dsx, it raises the question of what is the relationship between these two modes of regulation? It is proposed that regulation of fru by Dsx is the more ancient version of the sex determination pathway and that additional regulation of fru by Tra evolved subsequently, through the acquisition of regulatory RNA elements in the fru P1 transcript. This model is supported by studies of fru gene structures in distantly related Dipteran species, and species of other insect orders, that illustrate the considerable variability in the organization of sequences controlling fru splicing. Further, in some insects, no evidence for alternative splicing of fru has been found, yet fru still plays an important role in males to control courtship behaviors. Finally, in the Hawaiian picture-winged group of subgenus Drosophila, the fru orthologues lack the P1 promoter, and non-P1 fru transcripts exhibit male-specific expression, similar to what is proposed for non-P1 fru transcripts in D. melanogaster. Thus, it appears that regulation of fru by dsx may be the evolutionarily more ancient mechanism for sex-specific control of fru, while Tra-dependent splicing of P1 transcripts is a more recent adaptation. More broadly, tra is not conserved in the sex determination pathway in the majority of animal groups, while homologs of Dsx, the DMRTs, are virtually universal in animal sex determination. Thus, if Fru orthologs are involved in the creation of sexual dimorphism in the body or the brain in other animals, they cannot be regulated by Tra but may be regulated by DMRTs (Zhou, 2021).

Germ cells in Drosophila melanogaster need intrinsic factors along with somatic signals to activate proper sexual programs. A key factor for male germline sex determination is PHD finger protein 7 (Phf7), a histone reader expressed in the male germline that can trigger sex reversal in female germ cells and is also important for efficient spermatogenesis. This study found that the evolutionarily novel C-terminus in Phf7 is necessary to turn on the complete male program in the early germline of D. melanogaster, suggesting that this domain may have been uniquely acquired to regulate sexual differentiation. Genes were sought that regulated by Phf7 related to sex determination in the embryonic germline by transcriptome profiling of FACS-purified embryonic gonads. One of the genes positively-regulated by Phf7 in the embryonic germline was an HP1 family member, Heterochromatin Protein 1D3 chromoshadow domain (HP1D3csd). This gene is needed for Phf7 to induce male-like development in the female germline, indicating that HP1D3csd is an important factor acting downstream of Phf7 to regulate germline masculinization (Yang, 2021).

This study investigated how Phf7 regulates sex determination in the embryonic germline, and one of the interesting findings is that the unusual C-terminus of Phf7 is necessary for its effects in germline masculinization. The N-terminus of Phf7 is a conserved module comprised of three zinc fingers, of which at least one is functionally essential, and this part of the Phf7 protein evolved from G2E3 (G2/M E3 Ubiquitin Ligase), a protein also made up of three zinc fingers. In contrast, the C-terminus of Phf7 is evolutionarily novel and is not similar to any known domains, suggesting that this domain is undergoing very rapid evolution, a feature not uncommon for factors involved in sex determination (Yang, 2021).

Previously a phylogenetic analysis of Phf7 proteins was conducted across the species tree, and surprisingly it was found that Phf7 in insects and amniotes do not share a common ancestor. Those findings with the latest results indicate that Phf7 in these two animal branches are not orthologous to each other, and that the emergence of the novel C-terminus is likely a unique event that occurred in Drosophila to regulate sexual differentiation in the germline. Recently, mouse Phf7 was demonstrated to be expressed in spermatocytes and can ubiquitinate histones to facilitate histone to protamine exchange. This shows that the expression patterns and functions of D. melanogaster and mouse Phf7 are different, albeit both acting on the male germline. These observations further suggest that the C-terminus of D. melanogaster PHF7 evolved onto an existing module of three zinc fingers, thereby creating new ways to regulating germline sexual development. This is a very interesting example that adds to the collection of diverse mechanisms in sex determination (Yang, 2021).

What does this uncommon C-terminus of PHF7 do? The two most intuitive ideas are that it acts as a transactivation domain like those found in transcription factors, or that it can recruit other effector molecules through protein-protein interactions. The former idea did not hold up when tested in S2 cells. The possibility that the Phf7 C-terminus acts as a bridge between its histone-associating N-terminus and other transcription factors or chromatin factors to alter target gene expression is an appealing one but there is currently no direct data that support this idea (Yang, 2021).

Downstream effectors of Phf7 were sought in the embryonic germline, and it was revealed that HP1D3csd is activated by Phf7 to regulate germline masculinization. Two different genetic tests were perfomed, and while both indicated that Phf7 and HP1D3csd genetically interact, there were some differences in the results. In the Phf7-induced female germline loss assay, both reduction and gain of HP1D3csd expression were found to be exacerbated the Phf7-induced phenotype. In comparison, in the spermatogenesis rescue assay, loss of one HP1D3csd copy hampered rescue whereas HP1D3csd overexpression enhanced spermatogenesis in XX germ cells. The latter experiment is a more direct assay of germline masculinization whereas germline loss can potentially be caused by secondary effects unrelated to sexual development. Therefore, it is thought the results of the spermatogenesis rescue experiments more accurately reflect the relationship between Phf7 and HP1D3csd. In addition to the transcriptome results, HP1D3csd has been identified along with Phf7 to be a part of sex-biased mechanisms in other contexts not limited to the germline. These also support the model that Phf7 and HP1D3csd function synergistically (Yang, 2021).

Phf7 regulates male germline development, and it can associate with the active histone mark methylated H3K47, but it is unclear what Phf7 then does to regulate expression of target genetic loci. H3K9 methylation has also been reported to be important for maintaining sexual differentiation programs in the Drosophila germline. The identification of HP1D3csd as an important downstream factor provides interesting new ideas regarding how the male germline program is initiated and regulated. CSDs have been shown to interact with various chromatin remodelers, thus one appealing model would be that Phf7 can activate or even recruit HP1D3csd to loci important for germline masculinization. This would in turn bring chromatin remodelers to such genes for expression activation and regulation and initiate male-development of the germline. Given the other finding in this study that the C-terminus of Phf7 is an essential part of this process, it would be very interesting to now study which of these factors interact and cooperate with one another (Yang, 2021).

Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. Esg-interacting proteins were identified, and an interaction was demonstrated between Esg and the corepressor C-terminal binding protein (CtBP), which is also required for maintenance of hub cell fate. These results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo (Voog, 2014)

The homeostasis of self-renewal and differentiation in stem cells is strictly controlled by intrinsic signals and their niche. A large-scale RNA interference (RNAi) screen was conducted in Drosophila testes and 221 genes required for germline stem cell (GSC) maintenance or differentiation were identified. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in protein degradation pathway in cyst cells leads to testis tumor with overproliferated germ cells. Importantly, in the Cullin 4 - Ring E3 ubiquitin ligase (CRL4) complex, multiple proteins were identified that are critical to GSC self-renewal. pic/DDB1, the linker protein of CRL4, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice (Yu, 2016).

Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, a genome-wide transgenic RNAi screen was performed through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). This study identified 530 genes that regulate GSC maintenance and differentiation. Of these, 113 selected genes were further knocked down using cell-type-specific Gal4s, and more than half were found to be external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a Myb/SANT-like DNA-binding domain-containing heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. It was also found that mitosis/cytokinesis proteins are especially important for male GSC maintenance. These findings provide valuable insights and resources for studying stem cell regulation at the organismal level (Liu, 2016).

How tissues adapt to varying nutrient conditions is of fundamental importance for robust tissue homeostasis throughout the life of an organism, but the underlying mechanisms are poorly understood. This study shows that Drosophila testis responds to protein starvation by eliminating transit-amplifying spermatogonia (SG) while maintaining a reduced pool of actively proliferating germline stem cells (GSCs). During protein starvation, SG died in a manner that was mediated by the apoptosis of somatic cyst cells (CCs) that encapsulated SG and regulated their development. Strikingly, GSCs could not be maintained during protein starvation when CC-mediated SG death was inhibited, leading to an irreversible collapse of tissue homeostasis. The study proposes that the regulated elimination of transit-amplifying cells is essential to preserve stem cell function and tissue homeostasis during protein starvation (Yang, 2015).

Two broadly known characteristics of germ cells in many organisms are their development as a 'cyst' of interconnected cells and their high sensitivity to DNA damage. This study provides evidence that in the Drosophila testis, connectivity serves as a mechanism that confers to spermatogonia a high sensitivity to DNA damage. All spermatogonia within a cyst die synchronously even when only a subset of them exhibit detectable DNA damage. Mutants of the fusome, an organelle that is known to facilitate intracyst communication, compromise synchronous spermatogonial death and reduces overall germ cell death. These data indicate that a death-promoting signal is shared within the cyst, leading to death of the entire cyst. Taken together, it is proposed that intercellular connectivity supported by the fusome uniquely increases the sensitivity of the germline to DNA damage, thereby protecting the integrity of gamete genomes that are passed on to the next generation (Li, 2017).

Fatty acids are precursors of potent lipid signaling molecules. They are stored in membrane phospholipids and released by phospholipase A2 (PLA2). Lysophospholipid acyltransferases (ATs) oppose PLA2 by re-esterifying fatty acids into phospholipids, in a biochemical pathway known as the Lands Cycle. Drosophila Lands Cycle ATs oys and nes, as well as 7 predicted PLA2 genes, are expressed in the male reproductive tract. Oys and Nes are required for spermatid individualization. Individualization, which occurs after terminal differentiation, invests each spermatid in its own plasma membrane and removes the bulk of the cytoplasmic contents. This study developed a quantitative assay to measure individualization defects. Individualization is demonstrated to be sensitive to temperature and age but not to diet. Mutation of the cyclooxygenase Pxt, which metabolizes fatty acids to prostaglandins, also leads to individualization defects. In contrast, modulating phospholipid levels by mutation of the phosphatidylcholine lipase Swiss cheese (Sws) or the ethanolamine kinase Easily shocked (Eas) does not perturb individualization, nor does Sws overexpression. These results suggest that fatty acid derived signals such as prostaglandins, whose abundance is regulated by the Lands Cycle, are important regulators of spermatogenesis (Ben-David, 2015).

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. This study investigated caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. It was shown that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation (Aram, 2016). 

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown mitochondrial shaping roles were uncovered for other testis-expressed ATP synthase subunits. This study demonstrates the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern (Sawyer, 2017).

Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. This study shows that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex, localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp beta2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. These results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation (Lu, 2020).

Transcriptional regulation plays a central role in producing different cell types from the same genomic content. Throughout embryonic development, cells make and respond to cell fate decisions by turning on new transcription programs required to generate progressively more specialized cell types. Similar events drive differentiation of specialized cells from proliferating precursors in the adult stem cell lineages that maintain and repair many tissues throughout the life span. Understanding how cell type-specific transcription is achieved forms the very basis of understanding differentiation and development in multicellular organisms (Lu, 2020).

Tissue and stage-specific transcription programs are established by intricate interplay among promoter-proximal and distal DNA elements, and protein complexes that interact with them. Much recent work has focused on the role of stage or tissue-specific transcriptional activators and repressors acting upon distal enhancer elements to control the time and place of expression of developmental genes. However, evidence has emerged that variant forms of core promoter motifs and their recognition factors can play roles in cell type-specific transcription programs in certain tissues (Lu, 2020).

Several canonical core promoter motifs and promoter types have been identified and extensively studied. In Drosophila, TATA-box and/or downstream promoter element (DPE)-containing promoters tend to initiate transcription from a narrow region. The TATA box is bound by the TATA-binding protein (TBP), while the DPE is bound by certain TBP-associated factors (TAFs) in the general transcription factor TFIID to help precisely position RNA polymerase II for transcript initiation. On the other hand, promoters containing the DNA replication-related element (DRE) and/or other Ohler motifs (Ohler, 2002) tend to initiate transcription from a broad region and are thought to be associated with housekeeping genes. Recent work in Drosophila has shown that thousands of enhancers exhibit a distinct preference for one or the other of these promoter types (Zabidi, 2015; Rennie, 2018), suggesting that sequences near the transcription start site can play key roles in geneselective transcriptional regulation. However, the extent to which these canonical versus other core promoter motifs contribute to cell type-specific gene regulatory programs in differentiating cells and the molecular mechanisms by which they do so are not understood (Lu, 2020).

Male germ cell differentiation in Drosophila provides an excellent opportunity to study cell type-specific transcriptional regulation, as more than a thousand genes turn on for the first time in development when male germ cells become spermatocytes. In Drosophila, one germline stem cell normally produces a new stem cell and a gonialblast, which founds a clone of proliferating spermatogonia through four rounds of mitosis. The resulting 16 interconnected germ cells undergo a last round of DNA synthesis; then, as spermatocytes, they enter meiotic prophase. During this ~3-day period the spermatocytes express the vast majority of genes needed for later stages of male germ cell development. A recently developed heat-shock-Bam time course system (Kim, 2017) provided a way to obtain large quantities of germ cells at similar stages and greatly empowered study of the temporal events and molecular mechanisms that underlie the dramatic, cell type-specific change in transcriptional regulation that accompanies the transition from spermatogonia to spermatocyte (Lu, 2020).

Genetic and biochemical studies have identified two sets of cell type-specific proteins that regulate the spermatocyte transcription program, the tMAC complex (Beall, 2007) required for turning on most of the spermatocyte-specific gene expression program (Perezgasga, 2004; Doggett, 2011) and the testis-specific TAFs (tTAFs) required for full levels of expression of many genes in that program (Doggett, 2011; Lu, 2015). tMAC interacts physically with Achi/Vis, two highly similar TGIF-related TALE-class homeodomain proteins encoded by tandemly duplicated genes that are required for transcription in spermatocytes of tMAC-dependent genes (Ayyar, 2003; Wang, 2003). In flies null mutant for tMAC components, Achi/Vis, or tTAFs, germ cells arrest as mature spermatocytes. tMAC is a spermatocyte-specific version of the widely expressed and evolutionarily conserved MuvB core complex, which binds different protein partners, including members of the E2F, DP, Rb, and Myb protein families, to repress or activate key cell cycle and developmental genes (Sadasivam, 2013; Fischer, 2017). The tMAC complex expressed in Drosophila spermatocytes contains two proteins shared with the MuvB core: p55 Caf1 (RBBP4 in humans) and Mip40 (LIN37). tMAC also contains testis-specific paralogs of three of the other MuvB core components: Aly (paralog of Mip130 [LIN9]), Tomb (paralog of Mip120 [LIN54]), and Wuc (paralog of Lin52 [LIN52]). In addition, tMAC includes the testis-specific proteins Topi and Comr. Among the known tMAC subunits, Tomb, Topi, and Comr have predicted DNA-binding domains. Despite the importance of tMAC for turning on expression of most of the genes newly expressed in spermatocytes, the mechanism by which tMAC carries out this function is not known (Lu, 2020).

To investigate how the cell type-specific gene expression program for spermatocyte differentiation turns on, this study used RNA-seq to map transcript levels, CAGE to quantitatively map transcription start site (TSS) usage, and ATAC-seq to map chromatin accessibility as proliferating spermatogonia transition to differentiating spermatocytes. Combining these data, the promoters that turn on when germ cells become spermatocytes were shown to lack most of the canonical core promoter motifs. Instead, these promoters are enriched for the tMAC-ChIP motif and putative Achi/Vis-binding motif, and require tMAC function to become accessible and initiate active transcription as germ cells transition to the spermatocyte state. The findings from genome-wide analyses and selected reporter constructs are consistent with published results that regulatory elements sufficient for expression in spermatocytes lie close to the promoter regions in several genes expressed specifically in spermatocytes (Lu, 2020).

One of the striking findings from this study is that many genes expressed both in bam^-/- mutant testes, in which spermatogonia continue to proliferate, and in 72 hr post-heat shock testes, in which many germ cells have progressed to the spermatocyte state, use an alternate promoter that turns on only in the spermatocyte-containing sample. It is possible that the conditions for productive transcription initiation are so different in spermatocytes compared with spermatogonia that many genes evolved alternative promoters to allow transcription in both cell types. Use of promoters with distinct proximal motifs bound by cell type-specific promoter-interacting factors such as are describe in this study may contribute to down-regulation of the old program as well as to turning on a new cell type-specific differentiation program. Indeed, the DRE motif, enriched at broad promoters down-regulated when spermatogonia transition to spermatocytes, is bound by the DRE-binding factor protein DREF, which recruits the TBP paralog TRF2 to facilitate transcript initiation. Because DREF protein is expressed in spermatogonia but down-regulated as spermatocytes mature, promoters that depend on the DRE motif and DREF-TRF2 may no longer express efficiently in late spermatocyte stages (Lu, 2020).

Function of the testis-specific tMAC complex is required for the vast majority of both the off-to-on gene and alternative new spermatocyte-specific promoters to become open and accessible as spermatogonia transition to the spermatocyte state. Where a new alternative spermatocyte-specific promoter is located within the stretch of DNA that wraps around the -1 nucleosome of the old spermatogonial promoter, displacement of this -1 nucleosome is likely a prerequisite for the expression of the new promoter. Together with the requirement for tMAC function for opening and expression of the new alternative promoter, this suggests that tMAC may play a role in remodeling nucleosomes. It is not known whether tMAC opens local chromatin by binding DNA that is transiently detached from nucleosomes due to loosening or breathing of nucleosomal arrays or other chromosomal events, and then holding it in an open position, or whether tMAC can bind to DNA wrapped around nucleosomes, similar to a pioneer transcription factor. The tMAC subunit Comr has a winged helix domain, and certain winged helix domain proteins are able to bind DNA on one side and allow simultaneous histone binding. Although none of the core components of tMAC have as yet been shown to have nucleosome remodeling activity, genetic analysis in C. elegans suggests that the generally expressed MMB/dREAM components (SynMuvB genes in worms) can interact with and may recruit components of the nucleosome remodeling and histone deacetylase (NuRD) complex (Lu, 2020).

It is possible that many of the alternative new promoters arose as a by-product of an ability of tMAC to promote chromatin opening and/or transcription initiation at many sites in the genome. In fact, mechanisms have evolved to keep this propensity under restraint: Recent work has showen that action of the multiple zinc finger protein Kmg expressed in spermatocytes is required to limit activity of tMAC to its normal target genes. With loss of Kmg function, tMAC binds to many additional sites in the genome that are not previously annotated promoters, with some of these being activated for transcription initiation (Kim, 2007; Lu, 2020 and references therein).

These motif and TSS analyses suggest that the off-to-on genes that express from a narrow region of efficient transcription initiation differ from the canonical, previously described narrow promoters. Among the down-regulated genes, most of the promoters with a narrow 'region of efficient transcription initiation' also had narrow total span of cap analysis gene expression (CAGE) signals, likely a result of TFIID precisely positioning Pol II for transcription initiation. In contrast, most of the off-to-on genes with narrow regions of efficient transcript initiation had a wide total span of CAGE signals. In other words, although these off-to-on promoters had many usable and permissive TSS positions, they were dominantly expressed from just a few TSSs within a narrow region, likely facilitated by the upstream and downstream promoter motifs and Inr at optimal positions (Lu, 2020).

Traditionally, there has been much focus on the importance of distal enhancer elements rather than promoters in specifying cell type-specific transcription programs. This view, however, may be somewhat biased by the intense analysis of developmental regulatory genes like even-skipped, the cell cycle regulatory phosphatase string/cdc25, or the close-range developmental signaling molecule BMP5, which are expressed and function in several disparate places in the body. Because the regionally expressed transcriptional activators and repressors that combine to establish positional identity differ in different regions of the body, it stands to reason that a gene that is expressed in different specific places, such as Pitx1 in jaw, pituitary, and pelvis, would need to use different enhancer elements to specify activation in different regions, with the regulatory input from the different enhancers perhaps feeding in to a common generic promoter. The situation and constraints may be quite different for terminal differentiation genes that are only expressed in a single tissue. In this case, as is shown in this study for differentiation of male germ cells, it may be more possible for the key regulatory sequences that specify cell type-specific transcriptional activation to be built into the core promoter and promoter-proximal regulatory sequences (Lu, 2020).

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. This study reports the essential role of the germline linker histone dBigH1 during early spermatogenesis. These results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression (Carbonell, 2017).

Studies in Drosophila have provided important insights into the cellular pathways governing maintenance, proliferation, and differentiation of adult stem cell lineages, which is central to understanding normal tissue homeostasis and its alteration in disease. In particular, Drosophila spermatogenesis has become an ideal model system to study these questions. In the Drosophila testis, germ stem cells (GSCs) localize anterior, anchored to a niche of somatic cells (hub), and divide asymmetrically for self-renewal and to produce daughter progenitor gonialblast cells (GBs), which start the complex differentiation program that leads to the production of functional gametes. GBs are surrounded by 2 somatic cyst cells (Cs) and undergo four successive rounds of transit-amplifying (TA) mitoses with incomplete cytokinesis to produce a cyst of 16 sister spermatogonial cells that remain interconnected. Then, cysts differentiate to spermatocytes and undergo two meiotic divisions to produce 64 spermatids that develop to mature sperm cells (Carbonell, 2017).

bag-of-marbles (bam) is an important regulator of the first stages of spermatogenesis. Upon asymmetric division, daughter cells move away from the niche and escape Dpp/BMP-mediated repression. Bam expression increases during the first TA divisions, reaching a maximum at the 8-cell stage. Then, at the 16-cell stage, Bam expression decreases rapidly, TA proliferation stops, and differentiation into spermatocytes proceeds. How these crucial developmental transitions occurring during early male GSC lineage differentiation are regulated is not fully understood. Bam is a translational repressor that interacts with Bgcn (Benign gonial cell neoplasm) and Tut (tumorous testis) to repress Mei-P26 expression, establishing a regulatory feedback loop that governs spermatogonia proliferation. Bam also plays an important function in female oogenesis, in which it is repressed in GSCs by Dpp/BMP signaling and interacts with Bgcn to prevent translation of GSC maintenance factors (Carbonell, 2017).

This study report on the essential contribution of the Drosophila germline-specific linker histone H1 (dBigH1) to male GSC lineage development and differentiation. Linker H1s are intrinsic components of chromatin that interact with the nucleosome and regulate chromatin higher-order organization. In comparison to core histones, H1s are less well conserved, with most species containing several variants that play partially redundant functions. A conserved feature in metazoans is the presence of germline-specific variants that replace somatic H1s in germ cells (GCs). Vertebrates generally contain several male-specific variants (i.e., H1t, HILS1, and H1T2 in mice and humans) and one female-specific H1 (i.e., B4 in Xenopus and H1oo in mice and humans). In contrast, a single germline-specific linker histone dBigH1 exists in Drosophila, which is present in both the female and the male germline. Female-specific H1s are generally retained during early embryogenesis until zygotic genome activation (ZGA) . In this regard, in Drosophila, dBigH1 has been shown to maintain the zygotic genome silenced until ZGA is completed at cellularization, when dBigH1 is replaced by somatic dH1 (Carbonell, 2017 and references therein).

Little is known about the functions that germline-specific H1s play in GSC lineage development and differentiation. In mammals, h1t2 mutant mice show several abnormalities during spermatogenesis and have reduced fertility. Similarly, hils1 expression is reduced in men suffering from reduced sperm motility. However, h1t mutants do not show detectable abnormality or fertility defects. In females, H1oo is required for maturation of germinal-vesicle stage oocytes. Finally, in Caenorhabditis elegans, depletion of H1.1/HIS-24, which is abundant in the germline, affects GCs proliferation and differentiation and reduces fertility. This study shows that in Drosophila, dBigH1 is essential for male GSC lineage differentiation. dBigH1 and Bam form a double-repressor loop that regulates progression into spermatocyte differentiation. It study also shows that dBigH1 acts as a general repressor in spermatocytes and that dBigH1 is required for male GSC maintenance. Altogether, these results unveil the essential contribution of germline-specific linker histone H1 variants to GSC lineage development and differentiation (Carbonell, 2017).

This study shows that dBigH1 is required to silence bam, which is a master regulator of spermatogonia proliferation and differentiation. During the first three TA divisions, Mei-P26 facilitates accumulation of Bam, which reaches a maximum at the 8-cell stage. Then, Bam levels decrease and spermatogonia stop proliferation and differentiate to spermatocytes (see dBigH1 and Bam Form a Double-Repressor Loop that Regulates Entrance into Spermatocyte Differentiation). Several mechanisms are known to contribute to Bam downregulation after the 8-cell stage. High Bam levels downregulate Mei-P26 translation, establishing a regulatory feedback loop. In addition, several microRNAs have been shown to downregulate Bam translation. The current results suggest a model by which, in addition to translational regulation, dBigH1-mediated transcriptional repression is required to silence bam during spermatocyte differentiation. In the absence of dBigH1, bam is not silenced; thus, entrance to the spermatocyte differentiation program is blocked and spermatogonial cells accumulate. This accumulation is not accompanied by increased spermatogonia proliferation; since dBigH1 is absent during the TA divisions and, therefore, its depletion is not affecting Bam accumulation to reach the threshold that dictates proliferation stop. The current results indicate that in addition to bam, dBigH1 represses expression of multiple other genes in spermatocytes, suggesting that like in early embryogenesis, dBigH1 acts as a general silencing factor in spermatocytes. Altogether, these observations support a model by which dBigH1 acts after spermatogonia cease proliferation to set up the specific gene expression program that governs spermatocyte differentiation (Carbonell, 2017).

The results also show that Bam, which is an important translational repressor, downregulates dBigH1 expression during TA divisions. dBigH1 expression in TA cells decreases parallel to the progressive accumulation of Bam, being detectable in all 2-cell cysts and in some 4-cell cysts. It is not known whether Bam directly interacts with dBigH1 mRNAs. However, dBigH1 mRNAs are likely present during TA divisions, as they are detected before spermatocyte differentiation and bam-GAL4-induced dBigH1 depletion in TA cells reduces dBigH1 content, blocking spermatocyte differentiation. Moreover, Bam represses dBigH1 expression specifically in TA spermatogonial cells when it is driven by the ubiquitously active vasa promoter. Altogether, these observations suggest that Bam expression during the TA divisions inhibits dBigH1 mRNA translation. Later, when Bam levels decrease, dBigH1 translation resumes, reinforcing Bam downregulation through transcriptional silencing. The results show that this dBigH1/Bam double-repressor loop is crucial to license spermatogonia into spermatocyte differentiation. The important contribution of mechanisms that regulate mRNA translation during spermatogenesis has been extensively studied. However, the actual role of transcription regulation in these processes is not well understood. From this point of view, this work unveils a functional interaction during the early stages of spermatogenesis that integrates both translational and transcriptional regulation (Carbonell, 2017).

These results also suggest that dBigH1 is required for GSC maintenance, as shown by the strong developmental defects observed in nos > bigH1^RNAi testes in which dBigH1 depletion was induced in GSCs. These defects include the lack of testes in ∼10% of cases and the drastic loss of GCs in the rest of the affected testes. bam overexpression results in GSC loss. However, the contribution of dBigH1 to GSC maintenance is not likely reflecting a role in bam repression since, in knockdown nos > bigH1^RNAi testes, no derepression of a bam^P-GFP reporter was observed in vasa-positive hub-attached cells that showed no detectable dBigH1 expression. In this regard, it is known that bam is actively repressed in GSCs by the DNA binding proteins PMad/Medea that are downstream effectors of Dpp/BMP signals emanating from the somatic cells of the niche. Repression imposed by specific DNA binding proteins likely prevails over transcriptional silencing induced by general repressors such as dBigH1. dBigH1 expression is constrained to the primordial GSCs early in embryogenesis, being present in somatic cells as long as their transcriptional program is not turned on. In this scenario, it is tempting to speculate that dBigH1 is required in GSCs to repress the somatic gene expression program throughout development. In this regard, its replacement by somatic dH1 during TA divisions is particularly intriguing. How this replacement takes place and what the consequences of its misregulation are remain to be determined (Carbonell, 2017).

The presence of germline-specific histone H1 is conserved in metazoans. However, to date, detailed functional analysis of their contribution to germline development and differentiation was largely missing. From this point of view, this study unveils the fundamental functions that germline-specific linker histone H1 variants play in male GSC lineage differentiation, providing further understanding of the factors and mechanisms that regulate the dramatic developmental transitions associated with spermatogenesis (Carbonell, 2017).

The generation of reactive oxygen species (ROS) widely occurs in metabolic reactions and affects stem cell activity by participating in stem cell self-renewal. However, the mechanisms of transit-amplifying (TA) spermatogonial divisions mediated by oxidative stress are not fully understood. Through genetic manipulation of Drosophila testes, this study demonstrated that CG8005 regulated TA spermatogonial divisions and redox homeostasis. Using in vitro approaches, it was shown that the knockdown of CG8005 increased ROS levels in S2 cells; the induced ROS generation was inhibited by NAC and exacerbated by H(2)O(2) pretreatments. Furthermore, the silencing of CG8005 increased the mRNA expression of oxidation-promoting factors Keap1, GstD1, and Mal-A6 and decreased the mRNA expression of antioxidant factors cnc, Gclm, maf-S, ND-42, and ND-75. The functions of the antioxidant factor cnc, a key factor in the Keap1-cnc signaling pathway was further investigated; cnc mimicked the phenotype of CG8005 in both Drosophila testes and S2 cells. These results indicated that CG8005, together with cnc, controlled TA spermatogonial divisions by regulating oxidative stress in Drosophila (Chen, 2020).

Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recentl work has shown that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, this study reports that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging (Butsch, 2022).

In polyandrous internally fertilizing species, a multiply-mated female can use stored sperm from different males in a biased manner to fertilize her eggs. The female's ability to assess sperm quality and compatibility is essential for her reproductive success, and represents an important aspect of postcopulatory sexual selection. In Drosophila melanogaster, previous studies demonstrated that the female nervous system plays an active role in influencing progeny paternity proportion, and suggested a role for octopaminergic/tyraminergic Tdc2 neurons in this process. This study reports that inhibiting Tdc2 neuronal activity causes females to produce a higher-than-normal proportion of first-male progeny. This difference is not due to differences in sperm storage or release, but instead is attributable to the suppression of second-male sperm usage bias that normally occurs in control females. It was further shown that a subset of Tdc2 neurons innervating the female reproductive tract is largely responsible for the progeny proportion phenotype that is observed when Tdc2 neurons are inhibited globally. On the contrary, overactivation of Tdc2 neurons does not further affect sperm storage and release or progeny proportion. These results suggest that octopaminergic/tyraminergic signaling allows a multiply-mated female to bias sperm usage, and identify a new role for the female nervous system in postcopulatory sexual selection (Chen, 2022).

Membrane curvature recruits Bin-Amphiphysin-Rvs (BAR)-domain proteins and induces local F-actin assembly, which further modifies the membrane curvature and dynamics. The downstream molecular pathway in vivo is still unclear. This study shows that a tubular endomembrane scaffold supported by contractile actomyosin stabilizes the somatic cyst cell membrane folded around rigid spermatid heads during the final stages of sperm maturation in Drosophila testis. The structure resembles an actin "basket" covering the bundle of spermatid heads. Genetic analyses suggest that the actomyosin organization is nucleated exclusively by the formins - Diaphanous and Dishevelled Associated Activator of Morphogenesis (DAAM) - downstream of Rho1, which is recruited by the BAR-domain protein Amphiphysin. Actomyosin activity at the actin basket gathers the spermatid heads into a compact bundle and resists the somatic cell invasion by intruding spermatids. These observations reveal a distinct response mechanism of actin-membrane interactions, which generates a cell-adhesion-like strategy through active clamping (Kapoor, 2021).

Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. This study shows that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, these studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility (Praggastis, 2021).

Conservation of genetic toolkits in disparate phyla may help reveal commonalities in organ designs transcending their extreme anatomical disparities. A male accessory sexual organ in mammals, the prostate, for instance, is anatomically disparate from its analogous, phylogenetically distant counterpart - the male accessory gland (MAG) - in insects like Drosophila. It has not been ascertained if the anatomically disparate Drosophila MAG shares developmental parallels with those of the mammalian prostate. This study shows that the development of Drosophila mesoderm-derived MAG entails recruitment of similar genetic toolkits of tubular organs like that seen in endoderm-derived mammalian prostate. For instance, like mammalian prostate, Drosophila MAG morphogenesis is marked by recruitment of fibroblast growth factor receptor (FGFR) - a signalling pathway often seen recruited for tubulogenesis - starting early during its adepithelial genesis. A specialisation of the individual domains of the developing MAG tube, on the other hand, is marked by the expression of a posterior Hox gene transcription factor, Abd-B, while Hh-Dpp signalling marks its growth. Drosophila MAG, therefore, reveals the developmental design of a unitary bud-derived tube that appears to have been co-opted for the development of male accessory sexual organs across distant phylogeny and embryonic lineages (Kumari, 2021).

While the striking effects of seminal fluid proteins (SFPs) on females are fairly conserved among Diptera, most SFPs lack detectable homologues among the SFP repertoires of phylogenetically distant species. How such a rapidly changing proteome conserves functions across taxa is a fascinating question. However, this and other pivotal aspects of SFPs' evolution remain elusive because discoveries on these proteins have been mainly restricted to the model Drosophila melanogaster. This study provides an overview of the current knowledge on the inter-specific divergence of the SFP repertoire in Drosophila and compile the increasing amount of relevant genomic information from multiple species. Capitalizing on the accumulated knowledge in D. melanogaster, novel sets of high-confidence SFP candidates and transcription factors are presented, presumptively involved in regulating the expression of SFPs. This study also addresses open questions by performing comparative genomic analyses that failed to support the existence of many conserved SFPs shared by most dipterans and indicated that gene co-option is the most frequent mechanism accounting for the origin of Drosophila SFP-coding genes. It is hoped this update establishes a starting point to integrate further data and thus widen the understanding of the intricate evolution of these proteins (Hurtado, 2021).

Early work on de novo gene discovery in Drosophila was consistent with the idea that many such genes have male-biased patterns of expression, including a large number expressed in the testis. However, there has been little formal analysis of variation in the abundance and properties of de novo genes expressed in different tissues. This study investigated the population biology of recently evolved de novo genes expressed in the D. melanogaster accessory gland, a somatic male tissue that plays an important role in male and female fertility and the post mating response of females, using the same collection of inbred lines used previously to identify testis-expressed de novo genes, thus allowing for direct cross tissue comparisons of these genes in two tissues of male reproduction. Using RNA-seq data this study identified candidate de novo genes located in annotated intergenic and intronic sequence and determine the properties of these genes including chromosomal location, expression, abundance, and coding capacity. Generally, major differences were found between the tissues in terms of gene abundance and expression, though other properties such as transcript length and chromosomal distribution are more similar. Differences between regulatory mechanisms of de novo genes in the two tissues were also explored and how such differences may interact with selection to produce differences in D. melanogaster de novo genes expressed in the two tissues (Cridland, 2021).

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. CySCs were found to be maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, it was found that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. It is proposed that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline (Yuen, 2021).

The genitalia present some of the most rapidly evolving anatomical structures in the animal kingdom, possessing a variety of parts that can distinguish recently diverged species. In the Drosophila melanogaster group, the phallus is adorned with several processes, pointed outgrowths, that are similar in size and shape between species. However, the complex three-dimensional nature of the phallus can obscure the exact connection points of each process. Previous descriptions based upon adult morphology have primarily assigned phallic processes by their approximate positions in the phallus and have remained largely agnostic regarding their homology relationships. In the absence of clearly identified homology, it can be challenging to model when each structure first evolved. This study employed a comparative developmental analysis of these processes in eight members of the melanogaster species group to precisely identify the tissue from which each process forms. The results indicate that adult phallic processes arise from three pupal primordia in all species. In some cases the same primordia generate homologous structures whereas in other cases, different primordia produce phenotypically similar but remarkably non-homologous structures. This suggests that the same gene regulatory network may have been redeployed to different primordia to induce phenotypically similar traits. These results highlight how traits diversify and can be redeployed, even at short evolutionary scales (Rice, 2021).

The rapid evolution of seminal fluid proteins (SFPs) has been suggested to be driven by adaptations to postcopulatory sexual selection (e.g. sperm competition). However, it has been recently shown that most SFPs evolve rapidly under relaxed selective pressures. Given the role of SFPs in competition for fertilization phenotypes, like the ability to transfer and store sperm and the modulation of female receptivity and ovulation, the prevalence of selectively relaxed SFPs appears as a conundrum. One possible explanation is that selection on SFPs might be relaxed in terms of protein amino acid content, but adjustments of expression are essential for post-mating function. Interestingly, there is a general lack of systematic implementation of gene expression perturbation assays to monitor their effect on phenotypes related to sperm competition. This study successfully manipulated the expression of 16 SFP encoding genes using tissue-specific knockdowns (KDs) and determined the effect of these genes' perturbation on three important post-mating phenotypes: female refractoriness to remating, defensive (P1), and offensive (P2) sperm competitive abilities in Drosophila melanogaster. These analyses show that KDs of tested SFP genes do not affect female refractoriness to remating and P2, however, most gene KDs significantly decreased P1. Moreover, KDs of SFP genes that are selectively constrained in terms of protein-coding sequence evolution have lower P1 than KDs of genes evolving under relaxed selection. These results suggest a more predominant role, than previously acknowledged, of variation in gene expression than coding sequence changes on sperm competitive ability in D. melanogaster (Patlar, 2022).

The most studied pheromone in Drosophila melanogaster, cis-vaccenyl acetate (cVA), is synthesized in the male ejaculatory bulb and transferred to the female during copulation. Combined with other chemicals, cVA can modulate fly aggregation, courtship, mating and fighting. This study explored the mechanisms underlying both cVA biosynthesis and emission in males of two wild types and a pheromonal mutant line. The effects of ageing, adult social interaction, and maternally transmitted cVA and microbes - both associated with the egg chorion - on cVA biosynthesis and emission were measured. While ageing and genotype changed both biosynthesis and emission in similar ways, early developmental exposure to maternally transmitted cVA and microbes strongly decreased cVA emission but not the biosynthesis of this molecule. This indicates that the release - but not the biosynthesis - of this sex pheromone strongly depends on early developmental context. The mechanism by which the preimaginal effects occur is unknown, but reinforces the significance of development in determining adult physiology and behaviour (Cortot, 2022).

Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. This study employed the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. Known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, this study found that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane-associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. These data allow development of a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion (Wells, 2023).

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. This study observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPS^H355A, a point mutation that destroys the formation of cytoophidia, it was found that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPS^H355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, these data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells (You, 2023).

It will now be important to investigate whether any of the network genes is involved in loading or unloading SP from microcarriers or, indeed, whether they play a role in microcarrier assembly, particularly since they appear to be present in species where SP does not seem to be involved in signaling. The role of secondary cells in microcarrier morphology also needs to be examined in more detail. Furthermore, confirming that other SFPs identified in the proteomics analysis or main cell-expressed GPI-anchored proteins are microcarrier cargos should allow the functions of these structures to be assessed more extensively and may suggest molecular tools that could be used to screen for similar processes in higher organisms (Wainwright, 2021).

Sphingolipids are ubiquitous structural components of eukaryotic cell membranes which are vital for maintaining the integrity of cells. Alkaline ceramidase is a key enzyme in sphingolipid biosynthesis pathway; however, little is known about the role of the enzyme in the male reproductive system of Drosophila melanogaster. To investigate the impact of alkaline ceramidase (Dacer) on male Drosophila, Dacer deficiency mutants (MUs) were obtained and were found to displayed apparent defects in the testis's phenotype. To profile the molecular changes associated with this abnormal phenotype, de novo transcriptome analyses of the MU and wildtype (WT) testes was performed; 1239 upregulated genes and 1102 downregulated genes were obtained. Then, six upregulated genes (papilin [Ppn], croquemort [Crq], terribly reduced optic lobes [Trol], Laminin, Wunen-2, collagen type IV alpha 1 [Cg25C]) and three downregulated genes (mucin related 18B [Mur18B], rhomboid-7 [Rho-7], CG3168) were confirmed through quantitative real-time polymerase chain reaction in WT and MU samples. The differentially expressed genes were mainly associated with catalytic activity, oxidoreductase activity and transmembrane transporter activity, which significantly contributed to extracellular matrix-receptor interaction, fatty acids biosynthesis as well as glycine, serine, and threonine metabolism. The results highlight the importance of Dacer in the reproductive system of D. melanogaster and provide valuable resources to dig out the specific biological functions of Dacer in insect reproduction (Zhang, 2021).

Many ribosomal proteins (RPs) not only play essential roles in ribosome biogenesis, but also have "extraribosomal" functions in various cellular processes. RpL36 encodes ribosomal protein L36, a component of the 60S subunit of ribosomes in Drosophila melanogaster. This paper reports RpL36 is required for spermatogenesis in D. melanogaster. After showing the evolutionary conservation of RpL36 sequences in animals, it was shown that RpL36 expression level in fly testes was significantly higher than in ovaries. Knockdown RpL36 in fly testes resulted in a significantly decreased egg hatch rate when these males mated with wild-type females. Furthermore, 76.67% of the RpL36 knockdown fly testes were much smaller in comparison to controls. Immunofluorescence staining showed that in the RpL36 knockdown testis hub cell cluster was enlarged, while the number of germ cells, including germ stem cells, was reduced. Knockdown of RpL36 in fly testis caused much fewer or no mature sperms in seminal vesicles. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) signal was stronger in RpL36 knockdown fly testes than in the control testes, but the TUNEL-positive cells could not be stained by Vasa antibody, indicating that apoptotic cells are not germ cells. The percentage of pH3-positive cells among the Vasa-positive cells was significantly reduced. The expression of genes involved in cell death, cell cycle progression, and JAK/STAT signaling pathway was significantly changed by RpL36 knockdown in fly testes. These results suggest that RpL36 plays an important role in spermatogenesis, likely through JAK/STAT pathway, thus resulting in defects in cell-cycle progression and cell death in D. melanogaster testes (Fang, 2021).

The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. This study presents a previously unknown tissue-specific role of Ago in spermatid differentiation. A classical mutant of ago was identified that is semi-lethal and male-sterile. During the characterization of ago function in testis, ago was found to play role in spermatid development, following meiosis. Spermatogenesis defects was confirmed by silencing ago by RNAi in testes. The v mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, the subcellular localization of Ago protein was determined with mCherry-Ago transgene in spermatids. These findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology (Vedelek, 2021).

During spermatogenesis, the process in which sperm for fertilization are produced from germline cells, gene expression is spatiotemporally highly regulated. In Drosophila, successful expression of extremely large male fertility factor genes on Y-chromosome spanning some megabases due to their gigantic intron sizes is crucial for spermatogenesis. Expression of such extremely large genes must be challenging, but the molecular mechanism that allows it remains unknown. This study reports that a novel RNA-binding protein Maca, which contains two RNA-recognition motifs, is crucial for this process. maca null mutant male flies exhibited a failure in the spermatid individualization process during spermatogenesis, lacked mature sperm, and were completely sterile, while maca mutant female flies were fully fertile. Proteomics and transcriptome analyses revealed that both protein and mRNA abundance of the gigantic male fertility factor genes kl-2, kl-3, and kl-5 (kl genes) are significantly decreased, where the decreases of kl-2 are particularly dramatic, in maca mutant testes. Splicing of the kl-3 transcripts was also dysregulated in maca mutant testes. All these physiological and molecular phenotypes were rescued by a maca transgene in the maca mutant background. Furthermore, it was found that in the control genetic background, Maca is exclusively expressed in spermatocytes in testes and enriched at Y-loop A/C in the nucleus, where the kl-5 primary transcripts are localized. These data suggest that Maca increases transcription processivity, promotes successful splicing of gigantic introns, and/or protects transcripts from premature degradation, of the kl genes. This study identified a novel RNA-binding protein Maca that is crucial for successful expression of the gigantic male fertility factor genes, spermatogenesis, and male fertility (Zhu, 2021).

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process orb2 mRNAs and proteins are localized within the developing spermatid. To evaluate the role of orb2 mRNA 3'UTR in spermatogenesis, the CRISPR/Cas9 system was used to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNAs and proteins are not properly localized within the 64-cell spermatid cyst (Gilmutdinov, 2021).

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, testis single-cell RNA-sequencing (scRNA-seq) data was analyzed from two Drosophila melanogaster strains. Evidence was found that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. Low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 were found via scRNA-seq and RNA-FISH, and no evidence was found of germline nuclear roX1/2 localization. These results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in a database (Witt, 2021).

Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs (Lawlor, 2021).

METTL3, the enzyme that catalyzes the m(6)A RNA modification in Drosophila is highly conserved and essential in various eukaryotic organisms. Mettl3 and its homologs have been linked to biological processes such as gametogenesis. This study focused on characterizing the role of METTL3 in Drosophila spermatogenesis. This study used the Gal4-UAS system to ubiquitously knockdown Mettl3 in both somatic cyst cells and germline cells. Using immunostaining and confocal microscopy, spermatid bundles were found to mislocalize in testes that contain the morphologically abnormal swollen apical tip. These result suggests Mettl3 knockdown using TRiP.GL01126 results in spermatogenesis aberrations (Rowe, 2022).

Adult stem cells and their transit-amplifying progeny alter their proliferation rates to maintain tissue homeostasis. To test how the division rates of stem cells and transit-amplifying progeny affect tissue growth and differentiation, a computation strategy was developed that estimates the average cell-cycle lengths (lifespans) of germline stem cells and their progeny from fixed-tissue demography in the Drosophila testis. Analysis of the wild-type data using this method indicated that during the germline transit-amplification, the cellular lifespans extend by nearly 1.3-fold after the first division and shrink by about 2-folds after the second division. Cell-autonomous perturbations of the stem cell lifespan accordingly altered the lifespans of successive transit-amplifying stages. Remarkably, almost 2-fold alterations in the lifespans of stem cells and their immediate daughters did not affect the subsequent differentiation. The results indicate that the early germline division rates can adjust the following division rates and the onset of differentiation (Gadre, 2021).

The re-use of genes in new organs forms the base of many evolutionary novelties. A well-characterised case is the recruitment of the posterior spiracle. gene network to the Drosophila male genitalia. This study found that this network has also been co-opted to the testis mesoderm where is required for sperm liberation, providing an example of sequentially repeated developmental co-options. Associated to this co-option event, an evolutionary expression novelty appeared, the activation of the posterior segment determinant Engrailed to the anterior A8 segment controlled by common testis and spiracle regulatory elements. Enhancer deletion shows that A8 anterior Engrailed activation is not required for spiracle development but only necessary in the testis. This study presents an example of pre-adaptive developmental novelty: the activation of the Engrailed transcription factor in the anterior compartment of the A8 segment where, despite having no specific function, opens the possibility of this developmental factor acquiring one. It is proposed that recently co-opted networks become interlocked, so that any change to the network because of its function in one organ, will be mirrored by other organs even if it provides no selective advantage to them (Molina-Gil, 2023).

It has been observed that genes playing particular roles during organ development can be recruited to perform novel functions in other organs. The re-use, or co-option, of developmental genes in new organs, is the base of many evolutionary novelties. An example of this is the highly transparent crystallin proteins that refract light in the eye lens. In all vertebrates, α-crystallin evolved from the co-option of a small heat shock protein to the eye; while in birds δ-crystallin evolved from the co-option of a different protein, the Arginosuccinase lyase involved in arginine biosynthesis (Molina-Gil, 2023).

Although there is abundant research on single-gene co-option, few studies have considered the functional consequences of full-gene network co-option. One of the best-characterised co-option cases is the recruitment of the appendage-forming gene network to form the eye-spots that decorate butterfly wings in several species. Similarly, in the Drosophila melanogaster subgroup, the larval respiratory posterior spiracles and the adult male genitalia share the expression of numerous genes due to the recent co-option into the male genital disc primordium of a pre-existing gene network controlling the formation of the external larval respiratory organs. The co-option of this gene network to the male genitalia resulted in the formation of the posterior lobe, a structure present in D. simulans and D. mauritiana, closely related to Drosophila melanogaster, but not in the more distant D. biarmipes or D. ananassae species (Molina-Gil, 2023).

The formation of the posterior spiracles and the posterior lobes have been well-studied in D. melanogaster. Posterior spiracle organogenesis is regulated by a gene network activated in the eighth abdominal larval segment (A8) by the Hox protein Abdominal-B (Abd-B). The internal spiracular chamber is formed from A8 anterior compartment cells (A8a) when Abd-B activates in the dorsal ectoderm the transcription of the JAK/STAT signalling pathway ligand Unpaired (Upd) as well as the Empty spiracles (Ems) and the Cut (Ct) transcription factors. The external protruding stigmatophore is formed from both anterior and posterior compartment A8 cells when Abd-B activates the Spalt (Sal) transcription factor, which in turn activates engrailed (en) transcription in a unique A8 pattern. These primary factors activate the RhoGAP Cv-c and RhoGEF64C cytoskeletal regulators, the cell polarity gene crumbs (crb) and various Cadherins6 Abd-B also modulates the expression of wingless and the EGF regulator rhomboid genes generating A8 specific segmental information distinct from that in more anterior segments (Molina-Gil, 2023).

The posterior lobe is a hook-shaped structure used by the male to grasp the female during mating, which may act as a prezygotic reproductive isolation barrier facilitating speciation. In D. melanogaster, ten genes of the spiracle gene network are required for the formation of the posterior lobe, with their activation in at least seven cases being regulated in both structures by the same cis-regulatory elements (CRE). The study of two of these enhancers, revealed that the same DNA-binding sites activate the CRE's expression in both organs, making this one of the best-characterised cases of whole gene network co-option (Molina-Gil, 2023).

The recruitment of a gene network to a new organ exposes it to different selective pressures that may accelerate the appearance of novelties. One such novelty is the expression of the posterior segment determinant engrailed in the anterior compartment cells of the A8 segment. Engrailed is crucial during segmentation, and its expression in the anterior compartment is surprising given that En has been localised to the posterior cells all along arthropod evolution (Molina-Gil, 2023).

Drosophila segmentation results from the activation of the segment-polarity genes engrailed (en), hedgehog (hh) and wingless (wg) in periodic stripes of cells along the antero-posterior axis of the embryo. Once activated, the segment-polarity genes engage in cross-regulatory interactions that maintain their expression. As a result, En and its direct target hh become expressed throughout development in the posterior compartment of every segment, where they regulate cell tension and adhesive characteristics that prevent posterior cells from mixing with anterior compartment cells, generating stable signalling boundaries. Mutant embryos for either en or hh result in an almost complete fusion of segments. As the posterior spiracle is one of the few circumferential organs in the embryo, en activity in anterior A8 cells could be required for establishing the circumferential information pattern necessary for spiracle organogenesis that contributed to the evolution of the protruding posterior spiracles characteristic of dipteran larvae (Molina-Gil, 2023).

This study investigated when en was recruited to the anterior A8 cells (A8a) and the cis and trans regulatory elements responsible for it. A8a expression appeared in Diptera before the evolution of the posterior lobe, and evidence is presented that this is associated with a previous posterior spiracle gene network co-option event to the testis mesoderm. Working with Drosophila melanogaster, this study showed that Engrailed expression in the A8a compartment is not required for the spiracle's development and that the engrailed CRE controlling spiracle expression is required in the testis cyst cells for spermiation. This work presents an example of repeated sequential gene network co-option events involving tissues of different germ layers, and shows how this resulted in the generation of a bona fide pre-adaptive developmental expression novelty: the activation of the En transcription factor in the anterior compartment of the embryonic A8 segment where, despite having no specific function, it opens the possibility of this important developmental factor acquiring one in the future. The expression of en in the anterior compartment of the A8 segment, was likely caused by the regulatory interlocking of the co-opted networks. It is proposed that gene network interlocking occurs as the result of the use of the same gene network in several organs, so that any change to the network because of its functionality in one organ, will be mirrored in all organs even if it has no selective advantage in some of them (Molina-Gil, 2023).

Previous work showed that the ectodermally expressed posterior spiracle gene network had been co-opted to the ectodermal male genitalia in the Drosophila melanogaster clade. This study shows the same gene network has also been co-opted to the mesodermal head cyst cells (HCCs) in Drosophila. Part of the posterior spiracle gene network becomes activated in the HCCs using the same enhancers driving expression in the posterior spiracle (Molina-Gil, 2023).

Although Abd-B is expressed in the somatic testis cells during embryogenesis, it is not expressed in the adult HCCs. Two alternative explanations are suggested. The first possibility is that one of the spiracle primary targets, many of which encode transcription factors, becomes expressed in the testis HCC independently of Abd-B regulation, and this results in the activation of the other spiracle genes due to cross-regulatory network interactions. Alternatively, the embryonic Abd-B expression could be speculated to epigenetically modify the spiracle gene network leaving it in a poised state that later could become activated in the absence of Abd-B (Molina-Gil, 2023).

The finding that the posterior spiracle gene network has been co-opted twice to different organs, suggests that the coordinated activation of several transcription factors and signalling molecules of the network does not necessarily cause detrimental effects. The reported effects of experimentally inducing the ectopic activation of the eye gene regulatory network in D. melanogaster, a situation akin to what may happen during co-option, may help in understanding this. eyeless activation in the imaginal primordia results in the formation of ectopic eyes and causes morphological alterations leading to lethality. However, it has been noted that ectopic Eyeless expression in the wing only induces eye development in proximal cells where Dpp is also expressed, and similar results were observed with Hh43,44 showing that the activation of a gene network inducer does not cause developmental transformations in all cells where it is expressed. Thus, when the co-option of a gene network causes developmental transformations reducing the animal's fitness, it will be lost. However, if the co-option had no influence on local developmental processes, it could be tolerated giving the opportunity to the gene network elements to interact with allelic variants present in the population whose interaction could result in selective pressures fixing the trait. Such a series of steps may have led to the co-option of other developmental gene networks such as the activation of the appendage gene network in the butterfly wings that resulted in the formation of novel wing spot patterns (Molina-Gil, 2023).

Expression of Engrailed in posterior metameric stripes is characteristic of arthropods, but is also observed in Onycophorans and in certain worms indicating an ancient origin12,46,47. In flies, anterior En activation has rarely been reported48, and in D. melanogaster, anterior expression is the exception. En activation in A8a associated to the posterior spiracle is present in D. virilis, but not in E. balteatus, suggesting it originated in the higher Diptera (Brachicera). This is supported by analyses showing that in other Diptera like Bactrocera dorsalis (Tephritidae) and Lucilia sericata (Calliphoridae), that present well-developed larval posterior spiracles, engrailed expression is restricted to the posterior A8 segment. The A8a engrailed spiracle expression does not depend on the segmentation gene network, but is regulated by the posterior spiracle network through Abd-B, Sal and the JAK/STAT signalling pathway. Despite its complex regulation, this en enhancer has no function in Drosophila melanogaster's spiracle organogenesis. Taken together, Engrailed expression in the anterior compartment of A8 was acquired after functional posterior spiracles already existed in dipteran larvae and its origin had to do with testis evolution. Future experiments should establish if this occurred due to the de novo appearance of the enD CRE, or to a silent enD cryptic enhancer becoming activated due to the recruitment of a new transregulatory factor in the spiracles/testis gene network or to changes in chromatin accessibility (Molina-Gil, 2023).

Most insects have a pair of non-protruding spiracle openings in each trunk segment (see Interlocking of co-opted gene networks). In the hemimetabolous bug Oncopeltus fasciatus nymph or the holometabolous beetle Tribolium castaneum larva these are formed by an internal spiracular chamber that expresses the Ct protein,; however, in Oncopeltus, Ct expression is not regulated by Hox proteins as it is in Drosophila, but depends on the tracheal protein Trachealess. Although in Tribolium, Spalt is expressed in the lateral ectoderm, it is not associated with the spiracles. Similarly, Engrailed is restricted to a posterior stripe of cells in the segment that does not surround the spiracular opening. Analysis of E. balteatus embryos suggests Sal was recruited in Diptera to the protruding stigmatophore before engrailed was expressed in A8a. Analysis of Sal and En expression does not reveal any activation in E. balteatus testis, indicating the spiracle network is not expressed in the gonads of all Diptera. This situation changes in Drosophila species where both Sal and En are expressed in A8a associated with the spiracles as well as in the testes. The co-option of the posterior spiracle cascade to the testis and the recruitment of Engrailed expression may have been the result of the major sperm size and gonad morphology changes occurring at some point after the divergence of Episyrphus and Drosophila that could have required the selection of new genetic variants to maintain fertility. As the enD enhancer has no apparent function in the posterior spiracles, but is required for male spermiation in D. melanogaster, it is proposed that originally this CRE was selected for its testicular function and that expression in the posterior spiracles was a side effect caused by the interlocked regulation of the co-opted gene networks in both organs which was not eliminated because it had no deleterious effects. Finally, after the divergence from the virilis group, a second co-option event of the spiracle gene network in the male genital disc of D. melanogaster and closely related species led to the evolution of the posterior lobe. Given the extreme variability of the posterior lobe sizes between various D. melanogaster genotypes it is unclear whether the enD enhancer has a direct function on lobe development or if its expression there is aphenotypic, and only the result of its interlocking with other functional elements of the co-opted gene network (Molina-Gil, 2023).

These results suggest that the selection of a new trait in a co-opted gene network in one organ, results in its activation in all organs where the network is expressed. This implies a slower acquisition of new traits in co-opted gene networks as novelties would become discarded if they had detrimental effects in any of the interlocked organs. On the other hand, if the new trait (for example the activation of a novel transcription factor) was selected for its function in one organ, this would lead to the fixation of non-functional pre-adaptive traits with functional potential in all interlocked organs. Thus, it is proposed that co-option can lead to sensu stricto pre-adaptation cases, as opposed to the associated term of exaptation. While in exaptation the co-opted character had a previous selective function that has been recruited to perform a novel function (i.e., heat shock proteins recruited to form the eye crystallin, or feathers that could have served for heat regulation or sexual display before being co-opted for flight) cases like the expression of Engrailed in the posterior spiracle provide no selective advantage but could conceivably acquire it in the future, as has happened in the anterior wing of several Diptera where En has acquired a new role in wing pigmentation (Molina-Gil, 2023).

The regulatory architecture of gene expression is known to differ substantially between sexes in Drosophila, but most studies performed so far used whole-body data and only single crosses, which may have limited their scope to detect patterns that are robust across tissues and biological replicates. This study used allele-specific gene expression of parental and reciprocal hybrid crosses between 6 Drosophila melanogaster inbred lines to quantify cis- and trans-regulatory variation in heads and gonads of both sexes separately across 3 replicate crosses. The results suggest that female and male heads, as well as ovaries, have a similar regulatory architecture. On the other hand, testes display more and substantially different cis-regulatory effects, suggesting that sex differences in the regulatory architecture that have been previously observed may largely derive from testis-specific effects. The difference in cis-regulatory variation of genes across different levels of sex bias in gonads and heads was also examined. Consistent with the idea that intersex correlations constrain expression and can lead to sexual antagonism, this study found more cis variation in unbiased and moderately biased genes in heads. In ovaries, reduced cis variation is observed for male-biased genes, suggesting that cis variants acting on these genes in males do not lead to changes in ovary expression. Finally, the dominance patterns were examined of gene expression, and it was found that sex- and tissue-specific patterns of inheritance as well as trans-regulatory variation are highly variable across biological crosses, although these were performed in highly controlled experimental conditions. This highlights the importance of using various genetic backgrounds to infer generalizable patterns (Puixeu, 2023).

While the biochemistry of gene transcription has been well studied, understanding of how this process is organised in 3D within the intact nucleus is less well understood. This study investigated the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, super-resolution microscopy was used to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. It was found that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. Foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription (Ball, 2023).

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). This study showed that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. This study shows that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes (Layana, 2023).

Drosophila spermatogenesis is an ideal system to study the effects of changes in lipid composition, because spermatid elongation and individualization requires extensive membrane biosynthesis and remodelling. The bulk of transcriptional activity is completed with the entry of cysts into meiotic division, which makes post-meiotic stages of spermatogenesis very sensitive to even a small reduction in gene products. In this study, the effect of are described changes in lipid composition during spermatogenesis using a hypomorphic male sterile allele of the Drosophila CDP-DAG synthase (CdsA) gene. The CdsA mutant shows defects in spermatid individualization and enlargement of mitochondria and the axonemal sheath of the spermatids. Furthermore, it was possible to genetically rescue the male sterile phenotype by overexpressing Phosphatidylinositol synthase (dPIS) in a CdsA mutant background. The results of lipidomic and genetic analyses of the CdsA mutant highlight the importance of correct lipid composition during sperm development and show that phosphatidic acid levels are crucial in late stages of spermatogenesis (Laurinyecz, 2016).

Mammalian DEAD box helicase DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. This study reports phenotypes associated with a null Ddx1 (Ddx1^AX) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. This study demonstrates that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. These data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing (Germain, 2015).

Mutations in several genes cause failure to activate many genes in this transcription program and a meiotic arrest phenotype: mutant testes filled with mature primary spermatocytes that fail to enter the meiotic divisions or initiate spermatid differentiation. Molecular cloning and analysis revealed that proper activation of transcription of these terminal differentiation genes in spermatocytes depends on cooperative action of two classes of meiotic arrest genes, expressed specifically in spermatocytes, which encode homologs of either TBP-associated factors (tTAFs) or components of the testis meiotic arrest complex (tMAC), a testis-specific version of the mammalian MIP/dREAM and the C. elegans SynMuvB complexes. tMAC contains at least 3 potential DNA binding components, Comr, Topi and Tomb, as well as several subunits implicated in chromatin remodeling or performing structural roles within the complex. It has been suggested that the tMAC component aly may help remodel spermatocyte chromatin for global activation of the spermatocyte transcription program. Action of the tMAC complex is needed for transcription of the G2 cell cycle regulators Cyclin B, cdc25/twine and boule in spermatocytes and of a large set of spermatid differentiation genes, the tTAFs are required for full activation of the spermatid differentiation genes but are dispensable for expression of transcripts for the G2 cell cycle regulators (Lu, 2015).

Expression of both the tTAFs and testis-specific components of the tMAC complex is turned on in early spermatocytes but the two classes of genes do not depend on each other to be transcribed. Recruitment of the tTAF protein Spermatocyte arrest (Sa) to promoters of target spermatid differentiation genes required function of the tMAC component, Aly. Several additional meiotic arrest genes not directly involved in regulation of the spermatocyte transcription program have also been discovered (Lu, 2015).

This study shows that Mediator plays a key role in activating expression in spermatocytes of a large number of transcripts involved in meiotic cell cycle progression and spermatid differentiation. For several of the many Mediator subunits, spermatocyte specific RNAi knock down produced a range of meiosis I maturation arrest phenotypes in male germ cells. Knockdown of the mediator subunit Med22 by RNAi in spermatocytes resulted in a consistent meiotic arrest phenotype similar to the tTAF mutants, suggesting that Mediator may function with the tTAFs and tMAC to activate the transcription program for terminal differentiation. Expression of Drosophila Mediator complex components becomes upregulated in early spermatocytes just prior to expression of the tTAFs, and Mediator subunits colocalized with tTAFs in spermatocytes. Localization of Mediator subunits to chromatin in spermatocytes depended on tMAC but not tTAF function, while spermatocyte specific knockdown of Med22 by RNAi abolished localization of tTAFs to chromatin, suggesting that Med22 may recruit or stabilize tTAFs to chromatin for activation of transcription of differentiation genes. Consistent with this pathway, expression of most spermatid differentiation transcripts dependent on tMAC and the tTAFs also required function of Med22 in spermatocytes. Strikingly, expression of transcripts up regulated in early spermatocytes that did not depend on tMAC and the tTAFs remained largely unaffected in Med22 knockdown testes, suggesting that Mediator is not required for all activated transcription in spermatocytes, but mainly for turning on the developmentally controlled transcription program that depends on tMAC and tTAF. The Zn finger protein Topi, a component of tMAC, interacts structurally with Med22 and may recruit Mediator to target genes. These results suggest that Mediator serves as a key component in a gene regulatory cascade of transcription activation that establishes the expression program for terminal differentiation in the male germ line adult stem cell lineage (Lu, 2015).

Activation of cell-type-specific gene expression profiles underlies terminal differentiation programs in both embryonic and adult stem cell lineages. In many cases such stage- and cell-type-specific gene expression programs require cooperative action of sequence-specific transcriptional activators and tissue-specific components of the basal transcription machinery. Previous studies have demonstrated that full activation in spermatocytes of the transcription program for terminal differentiation of male gametes requires sequential action of the testis specific tMAC complex and five testis specific homologues of components of the general transcription factor complex TFIID (the tTAFs). How tMAC and the tTAFs function at promoters of differentiation genes is not yet understood at the molecular level although function of tMAC was needed to recruit the tTAF protein Sa to promoters of spermatid differentiation genes. The new data presented in this study indicate that subunits of the Mediator coactivator complex mediate the regulatory function of tMAC on the tTAFs, acting in a pathway to turn on robust expression of terminal differentiation genes in spermatocytes. Potentially functioning as a testis specific TFIID complex, the tTAFs are needed for full activation of transcription of hundreds of spermatid differentiation genes in primary spermatocytes but are dispensable for transcription of meiotic cell cycle genes activated in the same cells. It is possible that the gene selectivity of the tTAFs is determined primarily by structures of the promoters of differentiation genes. The general TAFs and TFIID were shown to confer promoter selectivity and facilitate transcription activation at promoters with no or less stringent TATA boxes in Drosophila systems. Alternatively, as the current results suggest, the gene selectivity of the tTAFs may be partly achieved through DNA sequence specific components in the tMAC complex. tMAC contains at least two potential sequence-specific DNA-binding proteins, Tomb and Topi. This study found that Topi and MED22 physically interact when coexpressed in S2 cells. The tTAF protein Sa failed to localize to meiotic chromatin without Med22 function, suggesting the tTAFs may be recruited or stabilized by Mediator at promoters of the spermatid differentiation genes. Similar coactivator cross-talk between Mediator and canonical TAFs was observed during activation of the metal response gene, MtnA, in Drosophila cell culture. Although TFIID and Mediator were recruited separately to the MtnA promoter, TFIID was only functional in the presence of Mediator (Lu, 2015).

Although expression and subcellular localization of Med22 protein proceeded protein accumulation and localization of Sa in spermatocytes, and the correct localization of Sa relied on function of Med22, the data do not prove direct recruitment of Sa by Med22. The tTAFs, including Sa, may be recruited by other mechanisms to promoters of differentiation genes, with function of Mediator needed to stabilize and facilitate assembly and function of the tTAF containing preinitiation complex. Despite that Sa and Mediator colocalized to both chromatin and the nucleolus in spermatocytes based on immunofluorescence staining, Sa protein was predominantly detected at the nucleolus, whereas the Mediator signal was more evenly distributed between chromatin and the nucleolus, again suggesting that Sa may not be directly recruited by Mediator, at least to the nucleolus. It is possible that once localized, Sa is stabilized through interaction with Mediator since both the nucleolar and chromatin localization of Sa was completely abolished in Med22RNAi. It was not possible to detect direct interaction between Mediator subunits and tTAF components in testis extracts. However, considering the limitation of the Drosophila in vivo system for such biochemical assays, the possibility cannot be ruled out that the two complexes physically interact in spermatocytes, since interactions between Mediator and TFIID have been demonstrated in vitro (Lu, 2015).

Of all the Mediator subunits this study attempted to knock down in spermatocytes by RNAi, knock downs of only a few caused the meiotic arrest phenotype. Of these, Med22RNAi is the only knockdown for which the meiotic chromatin resembled tTAF mutant chromatin. Phenotypic variations among different Mediator subunits were also observed in a previous study in SL2 cells, in which each of mediator subunit was knocked down by RNAi. The heterogeneity of Mediator subunit knockdown phenotypes may be a result of variation in RNAi efficiency. Indeed, analysis of protein by immunofluorescence revealed that the RNAi hairpin against Med26 that was tested did not knock down expression of the Med26 protein. Since the Gal4-UAS system was used to drive RNA hairpin expression in spermatocytes, it is worth noting that Mediator had been shown to be required for Gal4 mediated transcription at UAS-containing promoters. Therefore, Mediator subunits needed for structural integrity of the complex or directly involved in interaction with Gal4-AD might not give strong phenotypes due to the potential negative feedback on the RNAi process. Alternatively, the differences in RNAi knockdown phenotypes of individual subunits could reflect functional diversity of particular subunits (Lu, 2015).

Although previously thought to be a generally needed inert protein complex which passively bridge interactions between transcription activators and the general transcription machinery, including TFIID and PolII, recent studies have revealed a more active and even gene selective role for Mediator in transcription activation. Many Mediator subunits were found to selectively modulate and integrate regulatory signals of specific cellular and metabolic pathways. Previous studies also showed several Mediator subunits functioned as adaptors to bridge interactions of particular transcription factor(s) to the Mediator complex or subcomplexes. For example, the C-terminal domain and the activation domain of p53 specifically bind to the MED1 and MED17 subunits of Mediator, respectively. MED15 binds strongly to the activation domain of SREBP-1α to regulate lipid homeostasis, and MED23 binding to the ELK-1 activation domain is required specifically for adipogenesis. More interestingly, MED14 and MED25 mutants had opposite effects on cell size control in Arabidopsis, suggesting distinct Mediator subunits coexisting in the same cells can have distinct mechanistic roles in transcription regulation. The current work suggests Mediator is recruited in primary spermatocytes to target genes by the tissue- and cell type-specific transcription factor Topi through its physical interaction with MED22. Importantly, consistent with being recruited by sequence specific activators in the tMAC, Mediator is not generally required in spermatocytes for activating transcription, as most genes which did not require function of tMAC (as seen with aly^-/-) or tTAFs (as seen with sa^-/-), were expressed at relatively normal levels in Med22RNAi testes (Lu, 2015).

This study detected physical interaction only between MED22 and Topi, it is certainly possible that other putative sequence specific DNA binding factors in tMAC, such as Tomb, or peripheral to tMAC, such as Achi/Vis also recruit Mediator through specific interactions with other Mediator subunits. Topi and Achi/Vis were suggested to be both needed at promoters of most genes controlled by the aly class of meiotic arrest genes. In spermatocytes, signals from different components of tMAC instructing activation of transcription may be integrated by Mediator through interactions between tMAC subunits and specific Mediator subunits, before being transduced to the general transcription machinery containing the tTAFs. This study on how transcriptional activation signals maybe routed through Mediator to the general machinery in differentiating spermatocytes will also shed light on how master transcription factors and Mediator-enriched super-enhancers may interact with the basal machinery in a gene selective fashion (Lu, 2015).

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. This study shows that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, it was shown that Fest is required for proper execution of meiosis I (Baker, 2015).

This study shows that the developmental program of male gametogenesis imposes several levels of cell type- and stage-specific post-transcriptional control on expression of the key G2/M cell cycle regulatory component CycB during meiotic prophase and identifies two key developmentally regulated trans-acting factors involved. First, the cycB RNA expressed in spermatocytes has a short 3' UTR, only 130 nt long and missing previously identified translational regulatory sequences used in other cell types. Second, the RNA-binding protein Rbp4, expressed starting early in meiotic prophase soon after completion of pre-meiotic DNA synthesis, binds the short 3' UTR and blocks translation of cycB in immature spermatocytes. Third, the Rbp4-interacting protein Fest, also upregulated early in the spermatocyte period, is also required for blocking CycB expression in immature spermatocytes (Baker, 2015).

Rbp4 and Fest RNA and protein are expressed in very early spermatocytes prior to onset of transcription of cycB, which depends on action of the tMAC complex (White-Cooper, 1998; Beall 2007). As a result, when the cycB RNA is expressed, it arrives in a cytoplasm already primed for its proper cell type- and stage-specific translational repression. Expression of Cyclin B3 (Clb3) protein in budding yeast has been shown to be restricted to meiosis II via sequences in the CLB3 3' UTR that block translation during meiosis I. Although translational repression of CLB3 in meiosis I was important to prevent premature separation of sister chromatids, an event appropriate for meiosis II rather than meiosis I, trans-acting factors responsible for the stage-specific translational repression have yet to be identified. The current data reveal that translational repression of a cyclin (in this case CycB) is also a key feature of meiotic prophase during spermatogenesis in a metazoan animal. Surprisingly, expression of CycB in immature spermatocytes -- either in rbp4 or fest mutants or by a mutated CycB-eYFP reporter -- was insufficient to drive those cells immediately into meiotic division. This might be because action of the Cdc25 cell cycle phosphatase encoded by twine, which is also translationally repressed in immature spermatocytes and becomes translationally activated by the RNA-binding protein Boule only in mature spermatocytes, is also required to generate active Cdk1/CycB. (Baker, 2015)

One general model for Fest function invokes the possibility that Rbp4 recruits Fest to the cycB 3' UTR, where Fest is able to interfere with cycB translation. However, no compelling evidence was found of specific binding of Fest to the cycB 3' UTR in biotin pull-down experiments from testis extracts from flies expressing eYFP-Fest either with or without functional Rbp4, which suggests that either Fest is not recruited to the cycB 3' UTR, or that the biotin pull-down assay has limitations in detecting indirect RNA-protein interactions. As a result, it is important to consider other mechanisms for Fest function, including the possibility that binding of Fest to Rbp4 is needed only briefly to enact a post-translational modification of or conformational change within Rbp4 to promote its ability to recruit partners and/or repress translation. It is also technically possible that Fest and Rbp4 act in parallel pathways to regulate CycB. Finally, as the fest germ cell phenotype is dramatically stronger than that of rbp4, it is likely that Fest regulates other proteins in addition to Rbp4 (Baker, 2015)

It is not yet known how information about spermatocyte maturation is communicated to Rbp4 or Fest to allow translation of cycB in mature spermatocytes. One or more proteins could respond to input regarding cell size, given that spermatocytes grow 25-fold in volume during meiotic G2. Alternatively, given that translation of cycB in mature spermatocytes requires function of the testis TAF proteins, signals indicating the completion of the spermatocyte transcription program (not merely its onset) could trigger the reprieve from translational repression. Another possibility might be a meiotic arrest checkpoint mechanism triggered by transcriptional activity from unpaired chromatin, as seen in mammalian spermatocytes. Whatever the stimulus, it is clear that through stage-specific expression of the translational regulators Rbp4 and Fest in very early spermatocytes, the developmental program of male germ cell differentiation exerts additional layers of control over the core cell cycle machinery (Baker, 2015)

In the sexually reproductive organisms, gametes are produced by meiosis following a limited mitotic amplification. However, the intrinsic program switching cells from mitotic to meiotic cycle is unclear. Alternative polyadenylation (APA) is a highly conserved means of gene regulation and is achieved by the RNA 3'-processing machinery to generate diverse 3'UTR profiles. In Drosophila spermatogenesis, this study observed distinct profiles of transcriptome-wide 3'UTR between mitotic and meiotic cells. In mutant germ cells stuck in mitosis, 3'UTRs of hundreds of genes were consistently shifted. Remarkably, altering the levels of multiple 3'-processing factors disrupted germline's progression to meiosis, indicative of APA's active role in this transition. An RNA-binding protein (RBP) Tut could directly bind 3'UTRs of 3'-processing factors whose expressions were repressed in the presence of Tut-containing complex. Further, this RBP complex could execute the repression post-transcriptionally by recruiting CCR4/Twin of deadenylation complex. Thus, it is proposed that an RBP complex regulates the dynamic APA profile to promote the mitosis-to-meiosis transition (Shan, 2017).

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex (SC) formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the rDNA. Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. This study separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescent in situ hybridization (FISH) to early prophase I spermatocytes, it was shown that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, it was determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis while the autosomal-specific Teflon was dispensable. Overall, these results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction (Hylton, 2020).

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. This study has analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, these findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes (Kabakci, 2022a).

Meiosis is a key innovation that evolved before the eukaryotic radiation into the extant domain. The canonical program of this conserved process relies on meiotic recombination (MR). MR contributes to the initial pairing of homologous chromosomes and generates crossovers that maintain homologs linked as bivalent chromosomes until the onset of anaphase during the first meiotic division (M I). MR proceeds usually in concert with synapsis, which achieves close homolog pairing all along the chromosomes via formation of the synaptonemal complex (SC). In spite of the eminent significance of MR, diverse meiosis variants have evolved that do not rely on MR. A most thoroughly studied example of such an achiasmate meiosis occurs in Drosophila melanogaster. While meiosis is largely canonical in D. melanogaster females and includes MR, it is achiasmate in the heterogametic males. This sex-specific difference in meiosis is characteristic among higher dipterans. Its evolution is poorly understood, but may be linked to the suppression of recombination between sex chromosomes (Kabakci, 2022a).

In D. melanogaster spermatocytes, not only MR but also SC formation does not occur. Nevertheless, soon after the last spermatogonial mitosis, homologous chromosomes are paired all along their length, according to analyses with a lacO/lacI-GFP system and FISH. It remains to be clarified whether the pairing of homologous chromosomes in early spermatocytes during the S1 stage is driven by the same mechanisms that are responsible for the pervasive somatic homolog pairing in D. melanogaster. Importantly, the extensive pairing of homologs in spermatocytes lasts only a few hours. During the S2b/S3 stages, homolog pairing was no longer detectable at any of the analyzed 14 distinct locations with euchromatic lacO array insertions. Moreover, even sister chromatid cohesion appeared to be lost except at centromeres (Kabakci, 2022a).

The drastic loss of homolog pairing and sister cohesion in mid-stage spermatocytes starts concomitantly with the process of territory formation, which separates three major chromosome territories apart within the interphase nucleus. One of the major territories contains the chromosome (chr) 2 bivalent, another the chr3 bivalent and the third the chrXY bivalent. The additional bivalent of chr4, a small dot chromosome, is often associated with the chrXY territory. Territory formation breaks up all non-homologous associations between the large chromosomes. Such non-homologous associations are extensive in S1 spermatocytes. They arise from a coalescence of large blocks of pericentromeric heterochromatin into a chromocenter. Similarly, centromeres are clustered initially. Disrupting these non-homologous associations during territory formation at the S2b stage depends on condensin II activity and additional unidentified forces. Failure of territory formation leads to persistence of non-homologous associations until prometaphase I and consequential chromosome segregation errors (Kabakci, 2022a).

The mechanisms that break up non-homologous chromosome associations during territory formation disrupt also homolog pairing and sister chromatid cohesion, presumably because of inevitable side effects. However, normally, homolog separation does not proceed to completion already during spermatocyte maturation. Complete premature homolog separation is prevented by residual homolog conjunction maintained by a dedicated special system that serves as an alternative to canonical homolog linkage by crossovers. Large-scale mutant screening has led to the identification of three genes (tef, mnm, and snm) that are specifically required for this alternative homolog conjunction (AHC). A proteomic approach has recently uncovered an additional AHC gene (uno). Loss-of-function mutations in these four genes result in chromosome missegregation during M I, but exclusively in males. In mnm, snm and uno mutant males, both sex chromosomes and autosomes are distributed randomly during M I. In contrast, only autosomes are missegregated in tef mutant males during M I (Kabakci, 2022a).

The TEF protein includes three C2H2-type zinc fingers and is therefore predicted to bind to DNA. The SNM protein is a distant relative of the stromalins (SCC3/SA/STAG protein family). Stromalins are subunits of cohesins, complexes of crucial importance for chromosome organization during interphase and M phase in somatic and meiotic cells. However, SNM is not co-localized with core components of cohesin, indicating that it does not function as a cohesin subunit. MNM is encoded by one of many differentially spliced mRNAs transcribed from the highly complex mod(mdg4) locus. MNM has an N-terminal BTB/POZ motif that is shared among almost all of the more than 30 distinct protein products expressed from the mod(mdg4) locus. In addition, MNM has a unique C-terminal zinc finger motif of the FLYWCH type. These N- and C-terminal motifs of MNM are predicted to mediate protein-protein interactions. UNO does not have obvious similarities to functionally characterized proteins (Kabakci, 2022a).

MNM, SNM and UNO accumulate in early spermatocytes, eventually co-localizing during spermatocyte maturation in multiple subnucleolar foci. At the start of M I, these foci coalesce into a single prominent spot on the chrXY bivalent. In D. melanogaster, chrX and chrY are strongly heteromorphic, lacking extended euchromatic homology that could mediate specific pairing. However, both sex chromosomes harbor rDNA gene clusters in the centromere-proximal heterochromatin and these rDNA clusters function as pairing centers during male M I. The prominent dot formed by MNM, SNM and UNO on the chrXY bivalent at the start of M I is localized on the paired rDNA loci of chrX and chrY. Apart from the prominent dot on the chrXY pairing center, autosomal bivalents, which rely on euchromatic homology for pairing display far weaker dot signals of co-localized MNM, SNM and UNO. These autosomal dot signals were shown to be at least partially dependent on tef function. Strikingly, MNM, SNM and UNO disappear rapidly from all the bivalents within minutes during the onset of anaphase I. Separase, an endoprotease known to eliminate chromosomal cohesin at the metaphase to anaphase transition during mitotic and meiotic divisions, is required for the rapid disappearance of MNM, SNM and UNO from M I bivalents. UNO includes a separase cleavage site. Mutations that abolish this cleavage site prevent the rapid disappearance of MNM, SNM and UNO from M I bivalents and preclude homolog separation (Kabakci, 2022a).

The findings summarized above strongly support the notion that SNM, MNM and UNO function as proteinaceous glue that conjoins chromosomes into bivalents. However, it remains to be clarified how these proteins are recruited to chromosomes. SNM, MNM and UNO do not include known bona fide DNA-binding domains. They might therefore be recruited by other chromatin proteins. The zinc finger protein TEF is clearly an attractive candidate factor for chromosomal recruitment of the other AHC proteins. TEF's pattern of expression and its subcellular localization during spermatogenesis have not yet been characterized. This study closes this gap in understanding. Using transgenes encoding tagged functional versions of TEF, it was observed to be only transiently detectable in early spermatocytes. In contrast to the other known AHC proteins (MNM, SNM and UNO), TEF cannot be detected on bivalents at the start of M I, indicating that it is unlikely a stoichiometric component of the homolog-conjoining glue. However, evidence is provided that TEF can recruit MNM to chromosomes by direct protein-protein interaction. Indirectly, TEF can also recruit SNM-UNO, as they bind to MNM. Moreover, presumably by promoting AHC protein interactions, TEF stabilizes these proteins and controls their levels. AHC protein levels need to be controlled, as suggested by the consequences of simultaneous overexpression of all four AHC proteins in spermatocytes, which resulted in ectopic chromosome conjunction, failure of territory formation and segregation errors during M I. Ectopic expression of the four AHC proteins in somatic cells induced aberrant chromosome conjunction during mitosis, suggesting that AHC might not depend on additional spermatocyte-specific proteins beyond those already known (Kabakci, 2022a).

Genes required specifically for alternative homolog conjunction (AHC) during the achiasmate meiosis of Drosophila males were identified initially by extensive screening of mutants, and the teflon (tef) mutant phenotype was the first to be characterized in detail. The molecular identification of the affected gene revealed that tef encodes a zinc finger protein. This report provides more detailed functional characterization of the TEF protein. Expression pattern and intracellular localization during spermatogenesis were clarified with the help of tagged functional variants. An interaction between TEF and MNM was demonstrated by co-immunoprecipitation, and the responsible binding regions were mapped. Moreover, TEF was shown to bind to chromatin of polytene chromosomes in larval salivary glands. Importantly, TEF recruits MNM to chromatin, and via MNM also the other known AHC proteins SNM and UNO. Moreover, TEF potentiates the chromosome-linking activity of the AHC proteins SNM, UNO and MNM, as revealed by overexpression experiments in spermatocytes and other cell types (Kabakci, 2022a).

The TEF expression pattern was characterized with g-tef-sm_myc. This transgene under control of the tef regulatory region results in expression of a TEF version tagged with a spaghetti monster myc epitope tag (sm_myc). According to mutant rescue experiments, TEF-sm_myc is fully functional. Based on the g-tef-sm_myc expression pattern revealed by anti-Myc immunofluorescence, TEF is absent or low in somatic hub cells of testes but present in germline stem cells, spermatogonial cells and spermatocytes. TEF-sm_myc is also abundant in ovaries, where it is not germline-restricted as in testes. TEF's role in ovaries remains unclear, as no aberrant phenotype has been found in tef mutants so far (Kabakci, 2022a).

The subcellular localization of TEF-sm_myc was unexpected. During spermatogonial mitoses, a strong enrichment on centrosomes was observed. It is noted that mitotic centrosomal localization is also characteristic of CP190, an architectural chromatin protein, which like TEF has zinc fingers and interacts with a Mod(mdg4) protein. During interphase, TEF-sm_myc was primarily in many intranuclear foci of variable size, and a majority of these did not appear to be chromatin-associated. Intriguingly, the presence of TEF-sm_myc in spermatocytes was transient. TEF-sm_myc levels declined during spermatocyte maturation. It was no longer detectable in late spermatocytes (stages S5 and S6) and during the meiotic divisions. This subcellular localization and transient presence in spermatocytes were also observed in case of bamP-GAL4-VP16 driven UASt-tef-EGFP expression, which also rescues tef mutants. Importantly, in contrast to TEF, the other known AHC proteins (SNM, MNM and UNO, abbreviated as SUM) are all detectable on autosomal bivalents, when expressed analogously (as EGFP fusions from UASt transgenes with bamP-GAL4-VP16). These results argue strongly against the notion that homologous autosomes are conjoined by complexes of AHC proteins containing stoichiometric amounts of TEF. Rather than being an essential component of the glue that keeps homologous autosomes linked until onset of anaphase I, TEF might function only in early spermatocytes in the regulation of AHC establishment. It is noted that a presence of functional TEF in bivalents of late spermatocytes at levels below detectability is not excluded (Kabakci, 2022a).

Comparison of the mutant phenotypes caused by loss of tef, on the one hand, and loss of snm or mnm, on the other hand, provides further arguments against the notion that TEF is an essential component of the glue that conjoins autosomal homologs. The two tef alleles present in the transheterozygous mutants that have been analyzed are early non-sense mutations, shown to be amorphic with respect to meiotic chromosome transmission. However, the extent of autosome missegregation was significantly less severe in the transheterozygous tef mutants compared to snm and mnm mutants. This conclusion rests on concurrent findings made by time-lapse imaging of progression through M I and by analyses of meiotic chromosome missegregation with dodeca FISH. In snm and mnm mutants, bivalents are prematurely separated into independent univalents that are segregated randomly during M I. In contrast, in tef mutants there is some residual conjunction of autosomal homologs and their segregation is not completely random. Phenotypic comparisons are therefore consistent with the notion that TEF contributes to AHC establishment in early spermatocytes rather than also to the maintenance of AHC until anaphase I like the SUM protein. According to current observations after ectopic expression of AHC proteins, TEF might contribute to AHC establishment by promoting the recruitment of the SUM proteins to chromatin. TEF is the only AHC protein with a predicted bona fide DNA-binding domain. TEF has three zinc fingers, one in the N-terminal and two in the C-terminal region. Jointly, these N- and C-terminal zinc fingers mediate efficient TEF binding of TEF to polytene chromosomes after ectopic expression in larval salivary glands, as deletion of either the N- or the C-terminal region resulted in a substantial reduction of the chromosome-associated signals. Consistent with the absence of known DNA-binding motifs, none of the other AHC proteins displayed substantial binding to polytene chromosomes when expressed individually. Unexpectedly, however, polytene chromosome binding was clearly observed after co-expression of SNM and UNO. Presumably, these two proteins form a complex (SU) that includes a composite DNA-binding site. The two chromosome-binding entities among the AHC proteins, TEF and SU, have distinct preferences for chromosomal locations. However, both are able to recruit MNM onto polytene chromosomes. TEF and MNM interact directly according to co-immunoprecipitation experiments after transient expression in S2R+ cells, consistent with the previously observed co-purification of TEF with MNM-EGFP from testis extracts. The TEF-MNM interaction is mediated by the N-terminal part of TEF that includes the first zinc finger and by the C-terminal part of MNM. This C-terminal part is uniquely present in MNM. All the many additional isoforms that are generated by differential splicing from the complex mod(mdg4) locus have distinct C-terminal parts, and the three isoforms tested (T, C and P) were unable to bind to TEF. Beyond the TEF-MNM interaction, analyses of polytene chromosome binding and of co-immunoprecipitation suggested that all four AHC proteins (SNM, UNO, MNM and TEF) can co-assemble into SUMT complexes (Kabakci, 2022a).

Clearly, in salivary glands, TEF does not just bind to autosomes but also to the X chromosome. Thus, TEF does not appear to have an autosome-specific chromosome-binding ability that would explain why tef is required in spermatocytes for regular M I segregation of autosomes but not of sex chromosomes. It is suggested that the chromosome-specificity of the tef requirement might be linked to an additional effect of TEF on AHC proteins. According to the quantification of expression levels after Sgs3-GAL4-mediated expression of AHC proteins in salivary glands, formation of AHC protein complexes appears to stabilize these proteins. Levels of TEF and MNM were higher after co-expression compared to individual expression. Analogous observations were made with SNM and UNO. Similarly, after bam-GAL4-VP16-driven overexpression of SUM or SUMT in spermatocytes, the levels of the only tagged protein UNO-mCherry were increased by the presence of TEF. Moreover, MNM-EGFP levels were lower in tef mutant spermatocytes. Overall, these observations indicate a positive correlation between TEF and SUM protein levels. In tef mutants, some of the remaining SUM is presumably still recruited to autosomal bivalents due to the chromosome-binding activity of SU. However, as SUM levels during wild-type meiosis are far lower on autosomal bivalents compared to the chrXY bivalent, autosomal bivalents might be more strongly affected when SUM protein levels decrease as a result of a loss of tef function. TEF increases SUM protein levels presumably by promoting the formation of protein associations that are more stable than the individual proteins. Stimulating effects of TEF on SUM gene transcription are not excluded but unlikely as the analyses included experiments where the AHC proteins were expressed with exogenous regulatory sequences (UASGAL4 and hsp70) (Kabakci, 2022a).

The proposed explanation for the autosome-specific effect of tef mutations remains speculative, also because of the technical difficulties to detect SUM proteins on autosomal bivalents. Even the normal amounts of autosomal SUM proteins during wild-type meiosis are difficult to detect unequivocally and consistently in each spermatocyte. In this study, by analyzing fluorescent versions of UNO, a quantitative estimate is provided for the striking difference in the amount of SUM proteins on autosomes and sex chromosomes in normal spermatocytes. Around 25-100-fold lower amounts of UNO is found on autosomal bivalents compared to the chrXY bivalents. Without future technical improvements of detection sensitivity, a conclusive demonstration of the postulated residual autosomal SUM complexes in tef mutants is not feasible (Kabakci, 2022a).

If detectable, the autosomal SUM proteins appear to be confined to 1-2 dots per bivalent at NEBD I in normal spermatocytes. Do these dots mark the location of autosomal homolog conjunction, or might there be additional SUM complexes at other locations below the limit of detection that contribute to conjunction as well? Recent cytological analyses of meiotic quadrivalents in spermatocytes heterozygous for autosomal translocations have indicated that autosomal homolog conjunction is spatially constrained to dot-like chromosomal locations. The spatial control of autosomal homolog conjunction in spermatocytes appears to be analogous to that of canonical crossovers. As a rule, a single restricted region within the euchromatic portion of each autosomal chromosome arm is linked by AHC protein assemblies to its homologous region. Thus, AHC positions might be controlled by processes analogous to crossover interference. The particularly strong crossover interference in C. elegans has recently been proposed to involve spatially restricted biomolecular condensation of recombination nodule proteins in combination with a regulated coarsening process. It is tempting, therefore, to speculate about the significance of MNM's apparent liquid phase separation potential. Both MNM and TEF include substantial portions that are predicted to be intrinsically disordered. Such regions are thought to favor liquid-liquid unmixing when they confer multivalent interactions. At high levels of expression, MNM-EGFP formed droplets in salivary gland nuclei, while this was hardly observed with MNM-mCherry. The known weak dimerization of EGFP might reinforce multivalent associations. With TEF-EGFP droplets were not obtained when expressed alone but when co-expressed with MNM-mCherry, which was co-localized with TEF-EGFP in the droplets. Thus, droplet formation was stimulated by the EGFP tag when present on either MNM or its binding partner TEF. In case of untagged endogenous proteins, droplet formation might remain restricted to chromosomally recruited AHC assemblies. Accordingly, liquid phase separation of AHC proteins might be involved in the control of establishment or maintenance of alternative homolog conjunction in Drosophila spermatocytes (Kabakci, 2022a).

Experiments with spermatocytes revealed that an excess of AHC proteins is detrimental to regular chromosome segregation during male meiosis. Overexpression of SUMT severely inhibited chromosome territory formation most likely because it results in increased and more widespread conjunction between not only homologous but also non-homologous chromosomes. As a consequence, presumably, chromosomes fail to separate normally, often forming prominent bridges during anaphase and telophase of M I. The meiotic defects observed after SUMT overexpression are highly reminiscent of those caused by a loss of condensin II function. Conversely, absence of SUMT, as in mutants, has very similar phenotypic consequences as overexpression of the limiting condensin II subunit Cap-H2. Evidently, AHC proteins and condensin II have opposing activities that need to be in proper balance (Kabakci, 2022a).

The detrimental effects on meiotic chromosome segregation were much stronger after overexpression of SUMT compared to SUM. Overexpression of individual AHC proteins had barely any effect. These results provide further support for the proposal that in normal male meiosis, TEF assists in the chromosomal recruitment of SUM, the actual glue that maintains homolog linkage in bivalents until anaphase I onset. Accordingly, overexpression of TEF alone might not have severe detrimental effects because potentially low levels of endogenous SUM proteins might not allow excess assembly. Similarly, previous overexpression of individual SUM proteins was not observed to cause severe detrimental effects, perhaps also because low levels of other SUM subunits might limit excess assembly. The finding that ectopic SUMT expression in mitotically proliferating wing imaginal disc cells results in mitotic defects that resemble closely to the meiotic defects observed after SUMT overexpression in spermatocytes might indicate that AHC during male meiosis does not depend on additional spermatocyte-specific proteins beyond the known AHC proteins. The three proteins SNM, UNO, and MNM appear to be sufficient to induce conjunction between mitotic chromosomes and thus interfere with their normal segregation during anaphase. In combination with TEF, SUM had even more detrimental effects on mitotic chromosome segregation. However, much remains to be learned about the regulation that controls the appropriate chromosomal positioning of AHC during male meiosis (Kabakci, 2022a).

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. This study reports that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, it is suggested that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, this study demonstrates that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease (Kabakci, 2022b).

Drosophila male meiosis is achiasmate and therefore dependent on dedicated proteins (SNM (Stromalin 2), UNO (univalents only) and MNM (modifier of mdg4), together referred to as SUM, that maintain conjunction between homologous chromosomes in replacement for the missing crossovers. The main findings provide insight into the biochemical basis of (1) how the SUM proteins achieve this alternative homolog conjunction (AHC), and (2) how AHC is eliminated in time at the transition from metaphase to anaphase of M I to permit separation of the homologs to opposite spindle poles. In addition, our results are informative concerning the evolution of the AHC system (Kabakci, 2022b).

SNM and the C-terminal domain of UNO form a stable heterodimeric complex (SU_C). Based on sequence comparisons, AlphaFold structural predictions and XL-MS with recombinantly expressed and purified proteins, the SU_C complex is homologous to that formed by stromalin and the stromalin-binding region of α-kleisin. Stromalins and α-kleisins are components of cohesin complexes. While SNM was recognized as highly similar to stromalins early on, the very limited similarity of UNO to α-kleisins has escaped detection until now. The important and conserved N- and C-terminal domains of α-kleisins, which mediate its binding to the SMC heterodimer in cohesin, are not present in UNO. From an α-kleisin precursor, UNO has thus retained only the stromalin-binding region and the previously identified separase cleavage site (Kabakci, 2022b).

Stromalin, via positively charged surface patches, has recently been shown to promote DNA-binding of cohesin in vitro. Purified SU_C also binds DNA. At least one of stromalin’s positively charged surface patches [43] is clearly also present in SNM and contributes to the DNA-binding of SU_C, according to in vitro analysis with mutant versions of SU_C. In addition, a conspicuous, positively charged α-helix at the very C-terminus of UNO, which is absent from α-kleisins, makes a contribution to the DNA-binding of SU_C that is even more important than the basic SNM patch. Apart from DNA-binding, the interactions with the other AHC proteins were still normal in case of UNOchm-EGFP, a mutant with acidic or neutral residues in place of the six basic residues in the C-terminal α-helix. In vivo, UNOchm-EGFP displayed strongly reduced chromosome-binding and failed to provide normal AHC during male meiosis. These results strongly argue for the physiological importance of the DNA-binding activity of SU_C. It is speculated that the C-terminal α-helix of UNO might clamp down on a DNA double helix bound to the basic surface patches of SNM and thereby strongly increase the strength of DNA-binding. The binding of SU_C to DNA does not appear to be sequence specific. Clearly, in competition with the scrambled DNA sequence, this study has not detected increased binding to the 240 bp repeat sequence from the rDNA intergenic spacers, which appears to mediate sex chromosome conjunction (Kabakci, 2022b).

Beyond DNA, SU_C binds to MNM. Neither SNM nor UNO interact with MNM individually, indicating that prior association of SNM and UNO is required for MNM binding. These conclusions are based on co-immunoprecipitation experiments after transient expression in S2R+ cells. Of note, this study has not accomplished SUM complex formation with purified proteins in vitro so far. Attempts at expressing and purifying full length MNM were not successful. Moreover, the successfully purified C-terminal region of MNM (MNM_C), which mediates the binding to SU_C according to co-immunoprecipitation experiments, did not bind to SU_C in vitro. It is conceivable, therefore, that binding of MNM to SU depends on prior post-translational processing steps. At present, the inability to generate SUM complexes in vitro precludes a straightforward clarification of the issue whether SU can bind simultaneously to both MNM and DNA. However, the extended contacts between the C-terminal domain of UNO and SNM over long stretches provide ample space with interface potential, thereby increasing the likelihood of simultaneous binding of MNM and DNA to SU (Kabakci, 2022b).

MNM_C mediates binding not only to SU_C but also to TEF, as revealed by the co-immunoprecipitation experiments. The MNM-TEF interaction also remains to be re-constituted with purified proteins in vitro. However, in case of MNM_C, simultaneous binding of both TEF and SU_C is not feasible according to co-immunoprecipitation experiments (Kabakci, 2022b).

Beyond the interaction domains discussed above, analyses demonstrated the presence of multimerization domains in both UNO and MNM. It ia suggested that these domains are likely of crucial importance for the molecular mechanism whereby the SUM proteins generate AHC. In case of UNO, the N-terminal domain (UNO_N), which is highly conserved in UNO homologs, self-associates, forming dimers and tetramers when expressed and purified from bacteria. This UNO_N region has a predicted structure that is very distinct from that of the conserved N-terminal region of α-kleisins, indicating that the evolution of uno involved substitution of N-terminal in addition to deletion of C-terminal coding sequences in an ancestral α-kleisin gene. Multimerization in case of MNM is also mediated by the N-terminal region MNM_N. The primary sequence of MNM_N is identical to that of the N-terminal region present in the considerable number of alternative isoforms expressed from the complex mod(mdg4) locus. This common part of the Mod(mdg4) protein isoforms (thus also designated as Mod(mdg4)_CP) contains a BTB/POZ domain. This protein interaction domain present in many eukaryotic proteins with diverse functions has been shown to mediate homomeric dimerization. In addition, in case of the particular type of BTB domain that is also present in the Mod(mdg4) protein products, heteromeric and higher order multimerization has been reported based on SEC, native gel electrophoresis and crosslinking studies. These results confirm and extend these findings. Purified MNM_N/Mod(mdg4)_CP was found to form stable hexamers according to SEC-MALS. The hexamers were readily modeled by AlphaFold2 as a ring-like complex with three dimers, and negative-stain electron microscopy revealed ring-like complexes of an appropriate dimension. A recent preprint describing similar structural analyses of the same type of BTB domains (i.e. the TTK-type) derived from other Drosophila proteins (Lola and CG6765) and also from Mod(mdg4) provides further confirmation of the ring-shaped hexameric structure (Kabakci, 2022b).

Because of the multimerization domains (UNO_N and MNM_N/Mod(mdg4)_CP), the SUM proteins have presumably the potential to form extended protein assemblies that include many copies of the SU_C DNA-binding site. Thereby, they might be empowered to effectively and stably conjoin distinct double-stranded DNA molecules and function as a chromosome glue. Accordingly, AHC would not rely on a topological ring-like embrace as proposed to be provided by cohesin in case of sister chromatid cohesion. Since the tetramers and hexamers formed in vitro by purified UNO_N and MNM_N/Mod(mdg4)_CP are stable, SUM protein assemblies formed on bivalents in spermatocytes are expected to adopt a more solid rather than a liquid state. Indeed, FRAP analyses confirmed that the SUM proteins in the dots associated with the sex chromosome pairing regions do not undergo dynamic exchange (Kabakci, 2022b).

The proposed extended SUM protein assemblies with their multitude of DNA-binding sites are unlikely to conjoin exclusively homologous DNA strands. Presumably, sister DNA strands are connected as well (and perhaps even neighboring regions on the same strand). Previous characterizations of meiotic mutant phenotypes are consistent with this view. Absence of AHC function results in premature separation of bivalents into univalents in late spermatocytes and early in M I. The SOLO (Sisters on the loose) and SUNN (Sister unbound) proteins, which appear to function similar to the Rec8 cohesin complexes of other eukaryotes, still assure in these univalents a functional unification of sister centromeres for organization of a single kinetochore unit, as well as well as pericentromeric sister chromatid cohesion. In solo and sunn mutants, sister centromeres and pericentromeric regions lack cohesion, but bivalents are still present until the onset of anaphase I. As sister chromatid cohesion within the regions of chromosome arms is normally lost after territory formation already during spermatocyte maturation, the presence of bivalents in solo and sunn mutants during early M I suggests that the SUM proteins conjoin not just homologous chromatids but also sister chromatids. In support of this interpretation, snm solo double mutants display univalents during early M I. It is emphasizes that a chromosomal glue that conjoins DNA strands indiscriminately, as proposed for the SUM protein assemblies (i.e., sister strands and homologous strands in trans and perhaps also neighboring regions in cis) should be perfectly adequate if it is applied at the right time during spermatocyte maturation, i.e., after disruption of non-homologous chromosomal associations by territory formation but before complete disruption of homolog associations (Kabakci, 2022b).

Clearly, the proposal that AHC relies on extended assemblies of SUM proteins providing a high number of DNA-binding sites remains speculative and requires further investigation. For example, understanding how the formation of SUM protein assemblies is controlled and restricted to limited chromosomal regions will be crucial. The mechanism whereby SUM protein assemblies are targeted to the sex chromosome pairing rDNA loci on chromosome X and Y remains unexplained. In case of autosomal bivalents, TEF is likely involved in the initial establishment of SUM protein assemblies. However, after ectopic expression in larval salivary glands, TEF as well as SUM bind to a large number of polytene chromosome bands In contrast, in mature S6 spermatocytes, the autosomal SUM protein assemblies are spatially restricted to one or two dots per chromosome arm. Targeting of SUM protein assemblies to the sex chromosome pairing site and into autosomal dots might involve interactions with additional chromosomal proteins. Mod(mdg4)_T (also designated as 67.2 or 2.2), the most extensively characterized isoform expressed from the complex mod(mdg4) locus, interacts and co-operates with several chromatin architectural proteins (including CP190, HIPP1 and SuHw) at the gypsy insulator. Moreover, Mod(mdg4)_T in combinations with chromatin architectural proteins in various combinations is generally enriched at boundaries between topologically associated chromatin domains and also at button loci that promote the somatic pairing of homologous chromosomes. Recently, Mod(mdg4) function has been implicated in a striking example of chromosome pairing-dependent regulation of physiological gene expression. Thus, multimerization by Mod(mdg4)_CP is likely crucial for chromosomal associations other than AHC during male meiosis. Accordingly, by recruitment of the Mod(mdg4)_H isoform MNM for AHC, evolution might have co-opted a pre-adaption that achieves chromosomal associations by Mod(Mdg4)_CP multimerization (Kabakci, 2022b).

While Mod(mdg4) isoforms other than MNM were found to be unable of binding to SU, these other isoforms clearly have the potential to form heteromeric associations with MNM according to co-immunoprecipitation experiments. Moreover, based on yeast two-hybrid (Y2H) analyses, various other proteins with TTK-type BTB domains might also form heteromeric associations with MNM. Whether such heteromeric interactions are relevant of AHC remains to be clarified. However, phenotypic analyses with various mod(mdg4) alleles have argued against contributions to AHC by Mod(mdg4) isoforms other than MNM. Moreover, while heteromeric associations of Mod(mdg4)_T with other Mod(mdg4) isoforms can readily be detected by Y2H and co-immunoprecipitation after overexpression in S2 cells, their occurrence on chromosomes without overexpression is questionable according to chromatin-immunoprecipitation (Kabakci, 2022b).

Importantly, AHC must provide conjunction between homologs in bivalents that is very robust and yet also amenable to rapid and complete elimination after biorientation of all the bivalents in the M I spindle, so that homologs can be separated to opposite poles during anaphase I. Efficient destructibility of AHC was achieved by the evolutionary co-option of the α-kleisin-derived protein UNO. Like α-kleisin, UNO includes a separase cleavage site that is highly conserved among UNO orthologs. This cleavage site was shown to be required for AHC elimination and homolog separation during anaphase I. By exchanging the separase cleavage site in UNO with that cleaved by the bio-orthogonal TEV protease, this study provides evidence that UNO cleavage is indeed sufficient to eliminate AHC. In these experiments, TEV was expressed under control of cis-regulatory sequences from exu or betaTub85D in mid spermatocytes. The presence of normal chromosome territories and of a normal subcellular localization of UNOTEV-EGFP at the onset of TEV expression indicated that this TEV expression occurred after successful AHC establishment, which occurs early during spermatocyte maturation. However, as a consequence of TEV expression, bivalents were prematurely converted into univalents, as clearly indicated by cytological analyses and by time lapse imaging of progression into and through M I. UNO cleavage separates the multimerization domain UNO_N from UNO_C, which mediates DNA-binding in conjunction with SNM. Therefore, it is proposed that UNO cleavage dissociates the chromosomal SUM protein assemblies to an extent where the number of associated DNA-binding sites is no longer sufficient for tight linkage of distinct double-stranded DNA molecules. Clearly, alternative mechanisms of AHC elimination by UNO cleavage remain conceivable, and further work will be required to clarify the mechanistic details of AHC and its elimination (Kabakci, 2022b).

This study addressed the complex question of how complex structures are maintained after their initial specification by studying the Drosophila male stem cell niche, called the hub. Once specified, the hub cells need to maintain their position and architectural integrity through embryonic, larval and pupal stages of testis organogenesis and during adult life. The Hox gene Abd-B, in addition to its described role in male embryonic gonads, maintains the architecture and positioning of the larval hub from the germline by affecting integrin localization in the neighboring somatic cyst cells. The AbdB-Boss/Sev cascade affects integrin independent of Talin, while genetic interactions depict integrin as the central downstream player in this system. Focal adhesion and integrin-adaptor proteins within the somatic stem cells and cyst cells, such as Paxillin, Pinch and Vav, also contribute to proper hub integrity and positioning. During adult stages, hub positioning is controlled by Abd-B activity in the outer acto-myosin sheath, while Abd-B expression in adult spermatocytes exerts no effect on hub positioning and integrin localization. The data point at a cell- and stage-specific function of Abd-B and suggest that the occurrence of new cell types and cell interactions in the course of testis organogenesis made it necessary to adapt the whole system by reusing the same players for male stem cell niche positioning and integrity in an alternative manner (Schardt, 2015).

In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II filaments (Zipper) that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, which would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).

In wild-type testes, phalloidin staining reveals an F-actin-enriched contractile ring that encircles the spindle midzone (the bundle of interdigitated microtubules between the separating chromosome complements during anaphase and telophase) from late anaphase through telophase of both meiotic divisions. In contrast, in many meiotic divisions of chic mutants, no actin staining is visible at the cell equator. In most mutant ana-telophases with equatorial actin staining, only irregular patches of F-actin are observed. Chic protein is found in clear concentrations near the cell cortex, particularly in the equatorial zone. In chic mutants abnormal aggregations of F-actin are observed primarily in premeiotic mature spermatocytes at the S5 stage. These aggregates are enriched in alpha-spectrin and are almost invariably associated with ring canals, suggesting that these actin aggregates are in fact relatively undegraded remnants of the male fusome (see Drosophila Spectrin for more information on fusomes). It is suggested that in the absence of chic the disintegration of the fusome is partially blocked (Giansanti, 1998).

In addition to the absence of the contractile ring, the ana-telophases of all the chic mutants exhibit another obvious abnormality: a defect in central spindle morphology. In wild-type ana-telophases of both meiotic divisions, the two daughter nuclei are connected by a prominent bundle of interdigitating microtubules. In chic mutants, this central spindle structure is considerably less dense than in wild type, and microtubules show very little or no interdigitation. In wild type, the central spindle is already evident by mid-anaphase, before the actin ring constriction has caused substantial equatorial pinching. In the strongest chic mutants, these early stages of central spindle formation are never seen, indicating that the chic phenotype reflects a failure of central spindle assembly rather than a degradation of this structure in the absence of the actin ring. This result is surprising because the central spindle is a tubulin based cytoskeletal structure. There is substantial evidence that the central spindle is required for structuring of the actin based contractile ring but not similar evidence that the contractile ring is required for building the central spindle. Together, these observations indicate that chic mutations disrupt two major cytokinetic structures: the microtubule-based central spindle and the actomyosin contractile ring (Giansanti, 1998).

In wild-type primary spermatocytes during the prophase-prometaphase transition of the first meiotic division (stage M1), centrosomes migrate from a position just under the plasma membrane to the nuclear envelope, where they nucleate prominent asters. The two asters then separate and move around the periphery of the nuclear membrane, so as to establish a bipolar spindle. Similarly, during late telophase of the first meiotic division and the short interphase between meiosis I and meiosis II, asters separate and migrate to the opposite poles of secondary spermatocytes. Although the asters in chic mutants are improperly positioned by the start of prometaphase, relatively normal-looking bipolar spindles eventually form by late metaphase. It is remarkable that the function of these spindles, with respect to chromosome segregation, appears to be largely unimpaired. Only a very low frequency of onion-stage spermatids contain irregularly sized nuclei, such as micronuclei (Giansanti, 1998).

Lesions in twinstar (tsr), a gene encoding a Drosophila cofilin (an actin severing and depolymerizing protein), cause a syndrome of phenotypic effects that have both similarities and differences to those described above for chic mutations. In tsr, as in chic spermatocytes, centrosome separation and migration are defective, abnormal accumulations of F-actin are apparent, and cytokinesis often fails after one or both meiotic divisions. tsr and chic mutant phenotypes, however, can be easily distinguished on the basis of several criteria. In tsr mutants, the central spindle is normal, and the contractile ring still forms (though in misshapen and enlarged form), in contrast to the situation for chic. The types of F-actin aggregates formed in tsr spermatocytes are much different from those in chic. It is concluded that tsr and chic mutations differ in how they interfere with meiosis (Giansanti, 1998).

To further investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).

The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. It is of interest that these figures also show severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin , preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells. (Giansanti, 1998).

In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).

The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).

Membrane remodeling does not require endocytosis or exocytosis. Conventional endocytosis may not be important for movement of the cystic bulge, because FM1-43 staining of cell membrane demonstrates that membrane uptake does not take place around the actin cones, and blocking endocytosis using temperature shift of the shibire (dynamin) mutant does not affect cystic bulge movement. In addition, no concentration of alpha-adaptin has been observed in the region around the actin cones, suggesting that no coated pit formation occurs there. Conversely, clathrin mutants have defects in individualization, but the reason that individualization fails has not been well studied. The discrepancies in these data will only be resolved by further analysis of the clathrin mutant phenotype and studies of the effects of loss of function in other proteins in the endocytosis pathway (Noguchi, 2003).

Likewise, exocytosis may not play a major role in the membrane remodeling process, because membrane staining with the dye FM1-43 suggests that there is not a significant amount of membrane insertion at the sites around the actin cones, and treatment with the the exocytosis inhibitor BFA does not affect the movement of cystic bulge. These data do not completely exclude the possibility that exocytic events participate in remodeling, since the exocytosis could not be directly measured. However, it seems more likely that the large number of membrane invaginations that are present in the cystic bulge is a sufficient source of membrane to accomplish remodeling. The plasma membrane seems to be smoothly reorganized into thin tubular structures around the actin cones. Furthermore, ultrastructural observations have shown that the membrane around actin cones is flat, without any invaginating or docking membrane vesicles. These data support the idea that the cell membrane in the cystic bulge is directly deformed into a thin tubular structure (Noguchi, 2003).

Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. A tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin (CL) concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function has been generated earlier. This study shows that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A2, iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, it was shown that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA2 inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention (Malhotra, 2009).

The cardiolipin metabolism defect associated with Barth syndrome is manifested by the triad of CL depletion, monolyso-CL accumulation, and CL species diversification, i.e., the generation of CL molecules with different fatty acyl compositions. It is not clear whether the abnormal CL homeostasis actually plays a role in the pathogenesis of Barth syndrome, and if so, which aspect is the key factor. This study addresses this issue in a Drosophila model of Barth syndrome (Malhotra, 2009).

It was found that tafazzin deficiency in Drosophila, which alters CL homeostasis and reduces CL levels, also disrupts spermatid individualization during spermatogenesis, resulting in male sterility, and that this male-sterile phenotype can be suppressed by inactivation of the CL-degrading enzyme iPLA2-VIA, which partially restores CL homeostasis in double-mutant flies. These observations suggest that CL content, or at least the MLCL/CL ratio, plays a critical role in Drosophila spermatid individualization. It has been recently shown that the final stage of spermatid differentiation in Drosophila involves an apoptosis-like mechanism, in which the cytochrome c-dependent caspase activation is required for the elimination of excess cytoplasm. Cardiolipin has been shown to play important roles in mitochondria-dependent apoptosis and a recent report demonstrats that CL deficiency increases cells' resistance to apoptosis. Therefore, CL deficiency in Drosophila testes may prevent the syncytial spermatids from initiating the apoptosis-like mechanism required for normal spermatid individualization. (Malhotra, 2009).

The cardinal characteristics of Barth syndrome are cardioskeletal myopathy, exercise intolerance, neutropenia, abnormal mitochondria, and altered CL metabolism. Because in eukaryotes CL is localized exclusively in mitochondria and is required for optimal mitochondrial function, it has been generally assumed that the defective CL metabolism causes the pathophysiology of Barth syndrome. This study tested this hypothesis by genetically manipulating CL metabolism in the Drosophila model. It was found that partial restoration of CL homeostasis through genetic inactivation of iPLA2-VIA suppresses the male-sterile phenotype of tafazzin-deficient flies; this provides the direct evidence that altered CL metabolism is a major contributing factor in Barth syndrome (Malhotra, 2009).

The most abundant CL molecular species from various organisms and tissues contain only 1 or 2 types of fatty acids. In many mammalian tissues, the predominant fatty acyl moiety in CL is linoleic acid (C18:2). For example, 80% of CL molecules in heart and skeletal muscle are tetralinoleoyl CL. However, the role of CL molecular species in vivo remains speculative. The characteristic fatty acyl composition of CL in vivo is achieved through tafazzin-dependent remodeling of nascent CL. However, tafazzin deficiency, such as in Barth syndrome, results not only in abnormal CL acyl composition, but also in CL depletion and monolyso-CL accumulation. Thus, it is unclear which aspect of the CL metabolic disorder contributes to the pathogenesis of Barth syndrome. The finding in this study that the male-sterile phenotype of tafazzin-deficient flies can be suppressed by genetic inactivation of iPLA2-VIA, which prevents CL depletion and monolyso-CL accumulation without correcting the abnormal CL acyl composition, suggests that the abnormal levels of CL and/or monolyso-CL are important pathogenetic factors. Because a cardiolipin synthase mutant of yeast exhibits abnormal mitochondrial function, it is likely that the low CL content is critical in the molecular mechanism of Barth syndrome. Nevertheless, because Barth syndrome is a multisystem disorder, involvement of monolyso-CL accumulation and abnormal CL acyl composition may also play a role in certain tissues and organs (Malhotra, 2009).

The mature acyl chain composition of CL is achieved through a remodeling process, which requires the action of tafazzin. It has been previously shown that tafazzin catalyzes phospholipid-lysophospholipid transacylation that involves both deacylation of a phospholipid such as CL and reacylation of a monolyso-phospholipid, such as monolyso-CL. Unlike the CoA-dependent deacylation-reacylation cycle (Lands cycle), in which a nascent phospholipid is deacylated by a phospholipase A to yield a free fatty acid and a lysophospholipid that is then reacylated by an acyl-CoA-dependent acyltransferase, transacylation does not require acyl-CoA, and proceeds directly by transferring a fatty acyl chain from a phospholipid to a lysophospholipid; no phospholipase is involved and no free fatty acid is generated in the process. It was found that although the calcium-independent phospholipase A2, iPLA2-VIA, is not required for CL remodeling, in the absence of tafazzin, i.e., in the Barth syndrome model, the enzyme plays a major role in the depletion of CL and the accumulation of monolyso-CL (Malhotra, 2009).

The finding that the phenotypic features of tafazzin deficiency can be suppressed by inhibiting iPLA2-VIA activity identifies this enzyme as a potential target for therapeutic intervention in Barth syndrome. Indeed, it was found that treatment of cultured lymphoblasts from Barth patients with the iPLA2 inhibitor BEL partially restores CL homeostasis. The calcium-independent iPLA2-VIA has been implicated in a variety of biological processes, including phospholipid remodeling, arachidonic acid release, apoptosis, and store-operated calcium entry. In addition, iPLA2-VIA knockout mice develop age-dependent neurological impairment and mutations in the iPLA2-VIA gene have been identified in patients with infantile neuroaxonal dystrophy and neurodegeneration with iron accumulation in the brain. Therefore, a therapeutic approach to Barth syndrome based on the inhibition of iPLA2-VIA is likely to require either careful titration of the phospholipase inhibitor, or even its tissue-specific targeting (Malhotra, 2009).

Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, a male-sterile allele of the Drosophila homolog of human LIS1 was characterized. Centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, loss of attachments between the nucleus, basal body and mitochondria were observed. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. Dynein recruitment to the nuclear surface and spindle poles was shown to be severely reduced in Lis-1 male germ cells. It is proposed that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, since similar phenotypes were observed in flies null for Tctex-1, a dynein light chain. asunder (asun) was previously identified as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis (Anderson, 2009). It is now reported that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. A model is presented in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis (Sitaram, 2012).

Analysis of a hypomorphic, male-sterile allele of Lis-1 revealed that Lis-1 plays essential roles during Drosophila spermatogenesis. The data suggest that loss of dynein function is the root cause of the defects that were observe in Lis-1^k11702 testes, as mutation of the dynein light chain gene tctex-1 (Dynein light chain 90F) phenocopies mutation of Lis-1. Based on their overlapping phenotypes in male germ cells, genetic interaction, colocalization and co-immunoprecipitation, a model is presented in which Lis-1 and asun cooperate to regulate dynein localization during spermatogenesis (Sitaram, 2012).

These observations suggest that centrosomes of Lis-1 spermatocytes remain attached to the cell cortex and fail to migrate to the nuclear surface at entry into meiotic prophase. The phenotype of persistent cortical centrosomes during meiotic divisions has been characterized in abnormal spindles and nudE testes, and the presence of cortical centrosomes has been noted in Lis-1^k11702 metaphase spermatocytes in studies of nudE mutants. Dynein-dynactin and LIS-1 localize to the cell periphery in lower eukaryotes and cultured mammalian cells, as well as to the posterior cortex of Drosophila oocytes. However, no enrichment of dynein-dynactin or LIS-1 at the cortex of Drosophila spermatocytes has been detected. Cortical dynein has been implicated in regulation of mitotic spindle orientation in several systems, although the mechanism is not clear. These data suggest that dynein and LIS-1 are required in spermatocytes to release centrosomes from the cortex prior to meiotic entry (Sitaram, 2012).

Lis-1 spermatocytes exhibit free centrosomes, albeit at a much lower frequency than the phenotype of cortical centrosomes. Detachment of centrosomes from the cortex of primary spermatocytes is an earlier step in male meiosis than reassociation of the centrosomes with the nuclear surface at G2/M; hence, a failure of centrosomes to detach from the cortex is likely to mask a subsequent failure of nucleus-centrosome coupling. LIS-1 colocalizes with dynein-dynactin at the nuclear surface, and localization of dynein-dynactin to this site is severely impaired in Lis-1 spermatocytes and spermatids. Dynein-dynactin anchored at the nuclear surface has previously been implicated in mediating interactions between the nucleus and centrosomes during both mitotic and meiotic cell cycles. It is proposed that defects in nucleus-centrosome coupling in Lis-1 spermatocytes stem from disruption in localization of dynein-dynactin to the nuclear surface (Sitaram, 2012).

Previous studies in other systems concerning the role of LIS1 in dynein-dynactin recruitment to the nuclear surface have yielded conflicting results. In C. elegans embryos, dynein-dynactin was reported to localize normally to this site in the absence of Lis-1. In mammalian neural stem cells, however, Lis1 was shown to be required for recruitment of dynein to the nuclear surface at prophase entry. Similarly, it was observed that severe reduction of perinuclear dynein-dynactin in Drosophila Lis-1 spermatocytes at meiotic onset, suggesting that Lis-1 is required for this process. Conversely, normal levels of Drosophila LIS-1 were found at the nuclear surface of tctex-1 spermatocytes; thus, dynein-dynactin does not appear to be reciprocally required for LIS-1 recruitment to this site. This finding of reduced levels of dynein heavy chain on the nuclear surface of tctex-1 spermatocytes suggest that Tctex-1 light chain plays a specific role in localizing dynein complexes to the nuclear surface; alternatively, complex integrity may be compromised in tctex-1 mutants (Sitaram, 2012).

Previously reported has been the finding that asun regulates dynein localization during Drosophila spermatogenesis (Anderson, 2009). The characterization of the hypomorphic Lis-1^k11702 allele and the null asun⁹³ allele during Drosophila male meiosis reveals overlapping but distinct phenotypes. Lis-1^k11702 spermatocytes exhibit two classes of centrosome positioning defects: cortical (major phenotype) and free centrosomes (minor phenotype). By contrast, although most asun⁹³ spermatocytes have free centrosomes, they do not share with Lis-1^k11702 spermatocytes the phenotype of cortical centrosomes. These observations suggest that the role of asun in spermatocytes is limited to events at the nuclear surface, whereas Lis-1 additionally regulates cortical events. asun⁹³ spermatocytes undergo severe prophase arrest, possibly owing to failure of astral microtubules of free centrosomes to promote nuclear envelope breakdown. In Lis-1^k11702 spermatocytes, however, meiosis apparently progresses on schedule despite cortical positioning of centrosomes. The high percentage of asun⁹³ spermatids with increased numbers of variably sized nuclei, probably a consequence of cytokinesis and chromosome segregation defects, are also absent in Lis-1^k11702 testes. These observations suggest that spindle formation and normal progression through male meiosis require centrosomes to be anchored, either to the nuclear surface or the cortex (Sitaram, 2012).

Hypomorphic Lis-1^k11702 and null asun⁹³ round spermatids also show similarities and differences in their phenotypes. Both genes are required for recruitment of dynein-dynactin to the nuclear surface; this pool of dynein probably mediates nucleus-basal body and nucleus-Nebenkern attachments, which are defective in both mutants. Genes encoding Spag4 (a SUN protein), Yuri Gagarin (a coiled-coil protein) and GLD2 [a poly(A) polymerase] are required for nucleus-basal body coupling in spermatids, although it is not known whether they interact with ASUN or LIS-1 in this process. The current studies suggest that Lis-1, but not asun, is required for proper Nebenkern shaping and Nebenkern-basal body association; these functions might be mediated by dynein/microtubules acting at the Nebenkern surface. Nebenkerne are generated through fusion of mitochondria following Drosophila male meiosis. Two Nebenkerne bodies are occasionally present in Lis-1 and tctex-1 spermatids, implicating dynein in regulation of mitochondrial aggregation at this stage. Together, these observations suggest that the role of asun in spermatids is limited to events at the nuclear surface, whereas Lis-1 plays additional roles in regulating Nebenkerne (Sitaram, 2012).

Based on the studies of hypomorphic Lis-1^k11702 and null asun⁹³ mutant testes, a model is proposed in which LIS-1 is required for several dynein-mediated processes during Drosophila spermatogenesis, and ASUN is required for the subset of these processes that involve the nuclear surface. Both LIS-1 and ASUN promote recruitment of dynein-dynactin to the nuclear surface of spermatocytes and spermatids. The strong genetic interaction that was observe between Lis-1 and asun suggests that they cooperate in regulating dynein localization during spermatogenesis; the finding that LIS-1 accumulation on the nuclear surface is lost in asun male germ cells provides further support for this notion. The observed colocalization and coimmunoprecipitation of LIS-1 and ASUN suggest that they function within a shared complex to promote dynein-dynactin recruitment to the nuclear surface. Not interaction between Drosophila LIS-1 and ASUN proteins was detected by in vitro binding or yeast two-hybrid assays, suggesting that their association may be mediated by another protein(s) rather than being direct. Future studies on the nature of the ASUN-LIS-1 interaction should help elucidate the mechanism by which dynein-dynactin localizes to the nuclear surface during spermatogenesis (Sitaram, 2012).

Several proteins that promote dynein recruitment and centrosomal tethering to the nuclear surface have been identified. In C. elegans embryos, the KASH-domain protein ZYG-12, which localizes to the outer nuclear membrane and binds the inner nuclear membrane protein SUN-1, is required for these events. Another KASH-domain protein, Syne/Nesprin-1/2 (see Drosophila Nesprin), works in concert with SUN-1/2 to mediate nucleus-centrosome interactions during mammalian neuronal migration. Two additional pathways required for dynein recruitment to the nuclear surface at prophase have recently been identified in cultured mammalian cells. BicD2 binds dynein and anchors it to the nuclear envelope via its interaction with a nuclear pore complex protein, RanBP2. Similarly, CENP-F and NudE/EL act as a bridge between dynein and Nup133. It has not yet been determined whether mammalian LIS1 and ASUN function within these pathways or whether they act via a parallel mechanism to promote dynein recruitment to the nuclear surface (Sitaram, 2012).

The finding that a single copy of Lis-1^k11702 can drastically decrease the size of asun^f02815 testes suggests potential roles for Lis- 1 and asun in regulating division of male germline stem cells of Drosophila, as loss of cell proliferation can lead to reduction of testes size. Interestingly, Lis-1 has been reported to regulate germline stem cell renewal in Drosophila ovaries. Orientation of the cleavage plane during male germline stem cell division requires proper migration of centrosomes along the nuclear surface, and misorientation of the plane can lead to stem cell loss. Given the importance of Lis-1 and asun in mediating nucleus-centrosome coupling in Drosophila spermatocytes, it is possible that these genes also cooperate to regulate centrosomes during stem cell divisions in testes. In humans, the LIS1 gene is dose sensitive during brain development, as the disorder lissencephaly results from deletion or mutation of a single copy. Lis-1 spermatogenesis phenotypes reported in this study were observed in flies homozygous for a hypomorphic Lis-1 allele; flies carrying one copy of this allele displayed many of the same phenotypes but to a lesser degree. These findings suggest that precise regulation of LIS- 1 protein levels is essential for normal development in Drosophila. A requirement for Lis1 during spermatogenesis is conserved in mammals. Deletion of a testis-specific splicing variant of Lis1 in mice blocks spermiogenesis and prevents spermatid differentiation. LIS1 and dynein were shown to partially colocalize around wild-type spermatid nuclei, but dynein localization in Lis1 testes was not assessed. It remains to be determined if the functions of LIS1 in mammalian spermatogenesis are mediated through dynein and if the ASUN homolog regulates LIS1 localization in this system (Sitaram, 2012).

The disorganized symmetry of the axoneme and the isolated A-tubules at the distal end of the CPL suggest that each microtubule doublet grows asynchronously. Longitudinal sections often revealed isolated A-tubules ending in a moderately dense material at the tip of the axoneme that might be implicated in microtubule nucleation. The wall of the A-tubules may act as template for the B-tubules that, in turn, may support the growth of the C-blade that give continuity to the longitudinal sheets emerging from the C-tubules of the centriole. Similar conclusions come from structural analysis of procentriole formation in human cultured cells. However, the polymerization of B- and C-tubules is bidirectional in cultured cells, whereas B-tubules and C-blades elongate at their distal/plus ends in the Drosophila spermatid axoneme (Gottardo, 2013).

In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. This study reports that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. Cep290 was shown to be essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity; MKS proteins also delimit a centriole-independent compartment in mouse spermatids. These findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly (Basiri, 2014).

Compartmentalization of cellular processes is a critical aspect of cell biology. In many cases-such as in mitochondria and the ER-a semipermeable membrane surrounds an organelle to create a compositionally distinct compartment. In other cases, such as in the nucleus and cilium, the lumen of the organelle is continuous with the cytoplasm and compartmentalization is achieved by a specialized gate at its cytoplasmic interface. The ciliary gate is found in a region known as the transition zone at the base of the cilium. Within the transition zone, axonemal microtubules are crosslinked to the surrounding membrane and components of the ciliary gate mediate attachments between the axoneme, ciliary membrane, and centriole, giving the impression that the transition zone is a static and rigid structure (Basiri, 2014).

Aside from housing the ciliary gate, the transition zone is the site of multiple ciliary activities. As such, the transition zone is compositionally diverse and it is unclear which of its components comprise the ciliary gate. However, recent studies have suggested that genes mutated in the ciliopathies nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS) encode proteins that comprise two distinct modules essential for ciliary gate function (Basiri, 2014).

Centrioles and basal bodies, as well as cilia and flagella, are synonymous terms in different cellular contexts. For simplicity, this paper maintains the terms centriole and cilium throughout the text. Cilia can be divided into two types based on the location of the axoneme. In compartmentalized cilia, such as Drosophila sensory cilia, the entire axoneme is assembled and maintained within a membrane-bound compartment projecting from the transition zone at the distal end of a centriole. Cilia formed by compartmentalized ciliogenesis require intraflagellar transport (IFT) to move cargo through the transition zone, and IFT machinery directly interacts with transition zone proteins. Thus, the centriole, IFT, and the transition zone are generally essential for compartmentalized ciliogenesis (Basiri, 2014).

In cytoplasmic cilia, such as in mammalian and Drosophila spermatids and microgametes of the malarial parasite Plasmodium, the axoneme is found in the cytoplasm and the centriole is not attached to the cell or ciliary membrane. In Drosophila spermatids, the axoneme is initially composed of bare microtubules and later acquires its mature composition by incorporating proteins in the cytoplasm. Furthermore, IFT is not essential for cytoplasmic axoneme formation in Plasmodium microgametes and Drosophila spermatids. Thus, the separation of the centriole from a membrane-bound compartment and dispensability of IFT appear contradictory for a role of compartmentalization in cytoplasmic ciliogenesis (Basiri, 2014).

Interestingly, electron microscopy of spermatids in various insects including Drosophila identifies a membranous cap-like structure associated with the growing axoneme tip. This, structure, which is referred to as the ciliary cap, is morphologically similar to a typical compartmentalized cilium. Furthermore, mutations in candidate Drosophila transition zone proteins result in defects in spermatogenesis, and many MKS module genes are highly expressed in Drosophila testes. Together, these observations suggest that transition zone components-which are typically implicated in cilium compartmentalization-may also be involved in the formation of cytoplasmic cilia (Basiri, 2014).

This study provides insight into the mechanism by which transition zone proteins function in cytoplasmic ciliogenesis. MKS module proteins comprise a ciliary gate that migrates to compartmentalize the growing axoneme tip in Drosophila spermatids. These findings provide an example whereby the ciliary gate is dynamically associated with the surrounding ciliary architecture to sustain a compartment housing the site of axoneme assembly (Basiri, 2014).

In most species, ciliary gate function is dependent on both the ciliopathies nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS) modules, which exhibit some redundancy at the transition zone. However, mutation of MKS module genes in humans is associated with more-severe ciliary disease than is mutation of the NPHP module, suggesting that the MKS module plays a more basic role in cilium formation or function. Consistent with this, it was not possible to detect core genes of the NPHP module in Drosophila, implying that the MKS module is sufficient for cilium compartmentalization and that compartmentalization can be achieved by a relatively small subset of proteins (Basiri, 2014).

Classically, the transition zone has been regarded as a characteristic of the ciliary base that is restricted to the distal end of the centriole. Indeed, a mechanism involved in anchoring transition zone proteins to the centriole has been recently described. However, the current work demonstrates that the transition zone can function independently of the centriole and suggests that the transition zone, and not the centriole, is the determining factor in positioning the ciliary compartment along the axoneme. The assembly of the transition zone upon the distal ends of centrioles prior to cytoplasmic axoneme elongation in Drosophila spermatids suggests that initial transition zone assembly requires the centriole. However, as the axoneme elongates, this transition zone becomes spatially separated from the centriole and continuously migrates to compartmentalize the growing axoneme tip. The spatial separation between the centriole and the ciliary cap demonstrates that the transition zone can function free of centriolar association and while constantly migrating relative to the growing axoneme to create a centriole-independent ciliary compartment. Transition zone migration may also underlie centriole elongation. In normal spermatocytes, the centriole continues to elongate after the assembly of the transition zone upon its distal end. Because centrioles are not known to grow at their proximal ends, centriole elongation likely occurs by transition zone migration along the axoneme, followed by the expansion of centriolar proteins over freshly exposed axonemal microtubules. The excessive length distribution of centriolar proteins observed in cep290mecH spermatocytes may result from an impairment in the transition zone barrier, allowing centriolar proteins to aberrantly expand along the axoneme at the expense of the transition zone. This interpretation is consistent with the observation that transition zone size is decreased in cep290mecH (Basiri, 2014).

The base of the ciliary cap may contain additional proteins besides those of the MKS module. Previously, it was shown that two proteins, Uncoordinated and Chibby, are found simultaneously in both the centriole and at a distal focus, presumably the ciliary cap. Although the role of these proteins at the ciliary cap remains unclear, it is possible that they cooperate with the MKS module in ciliary cap compartmentalization. Furthermore, another structure associated with the base of the ciliary cap, referred to as the ring centriole, has been described in insects by electron microscopy. It was proposed that the ring centriole is homologous to a structure known as the annulus of mammalian sperm. However, unlike the transition zone, the ring centriole/annulus seems to be localized outside of the ciliary cap on the cytoplasmic side of its base and does not appear to contact the axoneme. Considering this, it is speculated that the ring centriole is a distinct structure from the transition zone and that MKS proteins are not part of the ring centriole but are instead components of the ciliary cap transition zone as in compartmentalized cilia. In support of this, this study observed that CEP290 and MKS1 do not precisely colocalize with Sept4 in mouse spermatids (Basiri, 2014).

In compartmentalized cilia of C. elegans, Chlamydomonas, and vertebrates, transition zone ultrastructure is characterized by features known as 'Y-links,' which are thought to mediate attachments between the axoneme and ciliary membrane. These Y-links are also thought to be involved in regulating entry into the ciliary compartment, and Y-link formation seems to be dependent on MKS protein function. Although Y-links have been observed in the transition zone of some insect sensory cilia, they have not been identified in Drosophila. Still, electron microscopy of Drosophila spermatids identifies vague links between the axoneme and the ciliary cap membrane, implying that structural connections between the axoneme and ciliary cap membrane are also present in migrating transition zones. Indeed, similar vague links are observed in analysis of both the Drosophila ciliary cap base and sensory cilium transition zone (Basiri, 2014).

Similar to Drosophila, mammalian spermatid axonemes also contain a segment that is exposed to the cytoplasm. Distal to this segment, the axoneme resembles a compartmentalized cilium and is closely associated with a membrane of distinct composition. However, it was unknown whether a transition zone defines these distinct axoneme segments in mammalian spermatids. The finding that CEP290 and MKS1 localize to the boundary of cytoplasmic and membrane-bound axoneme segments in mouse spermatids suggests that, similar to Drosophila, the distal, growing region of mammalian spermatid axonemes is also compartmentalized (Basiri, 2014).

Although axoneme compartmentalization is a defining characteristic of cilia, it has remained unclear if compartmentalization is essential for cilium formation or if it is instead required to satisfy ciliary functional requirements. The current observations suggest that compartmentalization is a conserved feature of ciliogenesis that is required for the integrity of axoneme assembly. Although the axoneme may be exposed to the cytoplasm after its initial formation, the process of axoneme assembly must occur within a distinct cellular environment. The transition zone may also play a direct role in axoneme assembly, and it has previously been proposed that the base of the ciliary cap confers 9-fold symmetry to the axoneme by organizing growing axonemal microtubules. This idea is supported by the observation that ciliary cap axonemes of cep290mecH are structurally destabilized (Basiri, 2014).

In summary, these findings demonstrate that the transition zone can migrate relative to the axoneme to sustain a compartment housing the site of axoneme assembly. It is proposed that axoneme compartmentalization is conserved as a prerequisite for proper cilium assembly and that the ciliary gate possesses a high degree of independence from the surrounding ciliary architecture to specify the location of the ciliary compartment along the axoneme (Basiri, 2014).

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM). In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis. This study shows that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction. The ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and the PCL does not resemble a typical centriole. Additionally, Poc1 is enriched at the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, it was found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila (Khire, 2017).

The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. This study shows that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. These results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells (Vieillard, 2016).

Cilia and flagella are highly conserved organelles involved in various cellular and physiological processes. Defects in cilia assembly are responsible for many human diseases. Cilia are built around a microtubule core, the axoneme, which grows from the basal body (BB), itself derived from the mother centriole. At the interface between the BB and the plasma membrane, a particular compartment, the transition zone (TZ), plays both structural and functional roles by regulating the traffic in and out of the cilium and by forming structural links between microtubules and the membrane. Several ciliopathies, such as the Joubert syndrome and the Meckel syndrome (MKS) or nephronophthisis (NPHP) are caused by defects in TZ components (Vieillard, 2016).

TZ assembly is a complex process starting with the docking of the mother centriole to cytoplasmic vesicles and the timely and spatially ordered assembly of multiple proteins. TZ components have been extensively described in several organisms. Genetic approaches in Caenorhabditis elegans and biochemical approaches in mammalian cells helped to establish a first comprehensive hierarchy of these components. They fall into different modules, namely MKS, NPHP, and CEP290. Proteins of the MKS module colocalize and together contribute to the formation of the TZ in various organisms, from C. elegans to mammals. In C. elegans, MKS, NPHP, and CEP290 cooperate to assemble the TZ. Upstream, MKS5 (mammalian Rpgrip1L or NPHP8) controls the assembly of the MKS and NPHP complexes on centrioles. However, variations exist between model organisms, and all TZ components are not conserved in all ciliated species. For example, MKS5 and several other NPHP proteins are absent from the Drosophila melanogaster genome. Additional components of the TZ have been identified, but their integration in the hierarchy of TZ assembly is not yet clear. For instance, Chibby (Cby) is a component of the TZ in Drosophila and vertebrates, but how Cby contributes to TZ assembly remains to be characterized. Cby is absent from C. elegans and protozoa genomes. Dila (Azi1 in mammals) is also not present in C. elegans but is required for TZ assembly in Drosophila or for ciliogenesis in mammals and is also associated with centriolar satellites in vertebrates. Therefore, key components of TZ assembly in C. elegans or mammals such as MKS5 are missing in Drosophila, and the mechanisms of TZ assembly in this organism are largely unknown (Vieillard, 2016).

Two main ciliated tissues are found in Drosophila: sensory neurons, in which cilia are required for transducing most senses, and sperm germ cells. Sensory cilia assembly relies on intraflagellar transport (IFT) and is hence compartmentalized in Drosophila. In contrast, flagella assembly in male germ cells is IFT independent and said to be cytosolic. Such cytosolic mode of assembly is also proposed in mammals for the formation of the sperm midpiece. In Drosophila, cytosolic flagella extension takes place inside a membrane ciliary cap, which requires Cep290 for proper organization and architecture. This membrane cap is built in G2 Drosophila spermatocytes, when all four replicated centrioles dock to the plasma membrane and grow a primary cilium. During meiosis, the four primary cilia are internalized together with centrioles, maintaining the membrane ciliary cap connected to the plasma membrane (Vieillard, 2016).

This study shows that MKS proteins are involved in Drosophila TZ assembly, but simultaneous removal of several MKS components only leads to very mild TZ and axonemal defects. Instead, deleting both Cby and Dila, two components of the TZ in Drosophila, leads to extremely severe TZ disorganization with complete loss of MKS components and severe reduction of Cep290. In absence of Cby and Dila, BBs fail to dock to the plasma membrane of spermatocytes and assemble the ciliary membrane cap required for sperm flagella elongation. In absence of this ciliary cap, aberrant growth of axonemal microtubules is observed in spermatocytes. This study has demonstrated that Klp59D, a kinesin-13 family member, is present at the ciliary cap and is required for proper and timely regulated growth of the axoneme. It is therefore propose dthat the ciliary cap is necessary to restrict and timely coordinate ciliary component assembly in Drosophila spermatocytes (Vieillard, 2016).

This study shows that TZ assembly in Drosophila involves cooperative actions of Cby and Dila, both in sensory neurons and male germ cells. More importantly, it is revealed that a timely control of the balance between membrane cap assembly by TZ components and microtubule elongation by kinesin-13 microtubule remodeling is required for proper axoneme formation in Drosophila male germ cells. (Vieillard, 2016).

Flagella assembly in Drosophila male germ cells does not rely on IFT, as null mutations in IFT components have no consequences on flagella formation. It is thus considered to be cytosolic, in contrast to the more widely conserved IFT-dependent ciliogenesis, which is said to be compartmentalized (Avidor-Reiss, 2015). However, proteins of the ring centriole/TZ such as Unc, Cby, or Cep290 are required to compartmentalize the flagellar growing end in Drosophila through the formation of a ciliary cap. The exact function of this ciliary cap is not clear (Avidor-Reiss, 2015). It was proposed that the formation of the ciliary cap is associated with the formation of a diffusion barrier, which protects the ciliary end from cytoplasmic proteins. The current results are in agreement with this hypothesis and suggest that the ciliary cap is required to create a specific environment that restricts axonemal growth, as precluding ciliary cap formation in dila81; cby1 mutants leads to aberrant microtubule extensions and unstable axonemes. On the other hand, the results show that unbalanced microtubule growth prevents ciliary cap formation. In agreement with this conclusion, previous work showed that treatment of spermatocytes with Taxol leads to Unc domain extension, associated with aberrant axoneme elongation and defective ciliary cap formation, when treatment was applied on spermatocytes before BB docking, but not when applied after BB docking (Riparbelli, 2013). Therefore, cytosolic ciliogenesis in Drosophila is regulated by a precise balance between microtubule extension from the centriole distal end and membrane cap formation (Vieillard, 2016).

This mechanism is apparently specific to cytosolic ciliogenesis. In dila81; cby1 sensory cilia, the total absence of the TZ is not associated with aberrant axoneme elongation. This also indicates that centriole docking in this tissue is a prerequisite to elongate the axoneme. Klp59D is not expressed in sensory neurons, and driving Klp59D shRNA expression in sensory neurons did not reveal any sensory defects. These differences could reflect particular properties of centrioles in Drosophila spermatocytes. Indeed, all four centrioles convert to BB in spermatocytes, whereas only the mother centriole does so in sensory neurons. Therefore, different sets of proteins should be involved in centriole maturation in spermatocytes compared with sensory neurons. Further work will be required to understand molecular differences between sperm and sensory basal bodies (Vieillard, 2016).

Another set of observations highlights the particular behavior of spermatocyte centrioles. Mutations in TZ proteins have shown that reducing the length of primary cilia-like/TZ in spermatocytes is associated with an increase in length of the centrioles. This was observed when removing Cep290, and this study made identical observations on B9d2, tctnΔ mutant testes. Thus, reducing ciliary cap extension allows centriole elongation, suggesting a balance in centriolar extension controlled by the TZ. Such a balance was also revealed by removal of Klp10A, which leads to an increase in centriole elongation and in a simultaneous reduction of primary cilia/TZ extension. Hence, the kinesin-13 proteins Klp10A and Klp59D likely play complementary functions at the spermatocyte TZ by restricting, respectively, centriole elongation or TZ elongation (Vieillard, 2016).

The function of kinesin-13 family members in ciliogenesis is still unclear and sometimes contradictory. In protozoa, such as Giardia intestinalis, Leishmania, or Trypanosoma, removal of kinesin-13 leads to longer flagella in agreement with a tubulin-depolymerizing function of these proteins. It has also been proposed that kinesin-13 proteins play a role in maintaining a free tubulin pool to promote axoneme assembly. However, recent studies suggest more complex roles of kinesin-13 members in cilia assembly. In mammals, Kif24 is required to regulate centriole length by interacting with CP110. Such a function is also true for Klp10A in Drosophila. Thus, one possible explanation for the observed phenotype in the dila81; cby1 double mutant would be that CP110 is prematurely removed from centrioles, as Dila and CP110 have strikingly similar localization inside the TZ lumen. However, complete removal or overexpression of CP110 in Drosophila did not lead to measurable ciliary defects and obviously did not lead to aberrant centriolar extensions in male germ cells. Therefore, modulation of CP110 activity cannot explain the aberrant axonemal growth observed in the testes in both dila81; cby1 mutants and Klp59D KD (Vieillard, 2016).

In Tetrahymena, kinesin-13 protein was shown to act as an axoneme assembly factor by regulating the levels of tubulin modification and in particular tubulin acetylation inside the cilia. It is tempting to speculate that Klp59D could play such a function in the ciliary cap of the sperm germ cell. However, it is difficult to test this hypothesis, as quantifications of differences in tubulin modifications inside the ciliary cap by IF are difficult and biochemical quantification of tubulin modifications is limited by the huge amount of nonaxonemal (cytoplasmic) modified microtubules in male germ cells (Vieillard, 2016).

This study reveals both analogies and differences in TZ assembly between Drosophila and other organisms. In mammals, MKS proteins play a critical function on TZ assembly and subsequently cilia function as revealed by human ciliopathies, in which these proteins are mutated. In C. elegans, mutations in the five core members of the MKS complex (Tctn, MKSR-1 [B9d1], MKSR-2 [B9d2], MKS1 [B9d3], and MKS6 [Cc2d2A]) have no effect on cilia architecture and sensory function. However, functional interactions were revealed between them, as their localization at the TZ is interdependent. In addition, whereas no genetic interactions are revealed when looking at sensory functions, combining mutations in several of these genes led to modified lifespan, suggesting that MKS components cooperate in specific ciliary signaling functions in C. elegans. In Drosophila, the current results indicate that, like in nematodes, the MKS complex has only subtle functions on TZ and cilia assembly and that MKS components exhibit functional interactions, as removing B9d2 and Tctn is sufficient to completely alter the recruitment of the other MKS members. In this context, genetic interactions are not expected to be revealed by combining other mutations in MKS components together with the B9d2, tctn mutant. The current observations therefore suggest that the hierarchy and function of MKS components are likely conserved between C. elegans and Drosophila (Vieillard, 2016).

In contrast, strong genetic interactions have been described between MKS and NPHP components in C. elegans. In particular, MKS5 (Rpgrip1L or NPHP8 in mammals) acts upstream of all other components to build the TZ, and NPHP4 and 1 interact with MKS complex proteins to organize the TZ. These NPHP members are not conserved in Drosophila. Therefore, other proteins should be required to organize the ciliary TZ upstream of the MKS complex in Drosophila. Among conserved candidates in other organisms, Cep290 is required to build the TZ in Drosophila, but MKS components are still assembled at the TZ in Cep290 mutants, thus suggesting that other TZ components act upstream of MKS. This study has demonstrated that cby and dila show strong genetic interaction in TZ assembly, whereas mutating each separately only shows moderate effects. These results highlight for the first time the synergistic roles of Cby and Dila in building the TZ (Vieillard, 2016).

Cby and Dila are not present in s but are conserved in mammals. In mouse, Cby plays an important role in motile ciliated epithelia, as demonstrated by the severe airway ciliogenesis defects observed in Cby knockout mice. In mammalian cells, Cby is also required for primary cilia assembly. Interestingly, mammalian Cby is required to dock centrioles to the plasma membrane, a function that is only revealed in Drosophila by removing both Cby and Dila. Mammalian Cby is required for Rab8-mediated ciliary vesicle assembly, and this study shows that Rab8 recruitment at the ciliary cap is also partially affected in cby1 mutant flies and strikingly completely abolished in dila81; cby1 double mutants. This suggests a conserved role of Cby in Rab8-associated membrane cap assembly from Drosophila to mammals and highlights that Dila is also required for this process in Drosophila. In mammals, Azi1 (Dila in Drosophila) is a centriolar satellite component required for centriole duplication, but it is also localized at centrioles and is required for cilia assembly. However, its function in TZ assembly has not been investigated. In Drosophila, Dila is found restricted to the BB and TZ and is required for cilia assembly, but no specific defects of the TZ were detected in mutant flies. Thus, this study in Drosophila reveals that Dila is also involved in building the TZ but that alternative pathways can compensate for Dila deficiency in TZ assembly. Such compensation mechanisms have also been proposed for Azi1 in mammals (Vieillard, 2016).

In conclusion, this study reveals critical genetic interactions between two yet-unrelated TZ proteins, Cby and Dila. Future work will be required to understand if such interactions are also conserved in mammals. This work also demonstrates that the formation of a ciliary cap is essential to coordinate ciliary assembly during cytosolic ciliogenesis by creating a biochemical environment that controls axonemal microtubule growth (Vieillard, 2016).

This study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins (Kotov, 2016).

This study shows that RNA helicase Belle (DDX3) is required cell-autonomously for the survival and divisions of GSCs in Drosophila testes. In bel6/neo30 mutants as well as in the case of germline-specific RNAi belKD rapid elimination of germ cells via apoptosis occurred. But what events could trigger apoptosis? To address this issue, larval testes were analyzed. Testes of bel6/neo30 mutant larvae still contained all populations of early germ cells. This observation indicates that primordial germ cells (PGCs) correctly migrate into embryonic gonads during mid to late embryogenesis. In the mutant larval testes the wild-type hub and GSCs adjacent to the hub were clearly detected. It is known that the mechanism of capturing GSCs and CySCs to the hub involves a high level of adhesion molecule E-Cad on the hub/stem cells interface. In testes with STAT depletion the expression of E-Cad is severely disrupted accounting for the defects in hub-GSC adhesion and for the subsequent loss of GSCs. However, it was determined that STAT expression (in CySCs) and consequently upstream Upd signaling from the hub were not disrupted in the bel6/neo30 testes. Although the amount of Belle was strongly reduced in the bel6/neo30 testes, no reduction of E-Cad level was observed in CySCs. On the contrary, high ectopic expression of E-Cad was detected on the surface of CySCs surrounding the hub. The influence of Belle on the adherens junction formation in GSCs cannot be directly estimated. However, due to adhesion failures a loss of GSCs via premature differentiation could be expected followed by normal development of newly formed germline cysts. In contrast, this study detected a reduced germ cell content and morphological abnormalities of early germ cells including their giant nuclear and cellular sizes. It is assumed that these germ cells could not enter mitosis and are delayed in the G2 phase. Failure to enter mitosis after G2 delay appears to induce germ cell apoptosis in the bel testes, as previously has been shown for the how testes (Kotov, 2016).

It is known that Drosophila mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, each form complexes with Cdc2, and they appear to function synergistically to provide a progression throughout mitosis. Sufficient levels of mitotic cyclins must be accumulated at the end of G2 to ensure the onset of mitosis. To date, cell cycle regulation of GSCs and their daughter gonial cells is still poorly understood. It is known that PGCs suppress mitotic activity during their migration to embryonic gonads due to translational repression of maternal cycB mRNA via its 3'UTR by Pumilio-Nanos complex and other unidentified factors. Pumilio and Nanos are also known to be expressed in GSCs of gonads of adult flies and are found to be essential for GSC maintenance. However, factors overriding the repressive Pumilio-Nanos-dependent signal and providing expression of zygotic Cyclin B protein during normal testis development are currently unknown (Kotov, 2016).

It has been shown that Cyclin B and Cyclin A, but not Cyclin B3, are required in the gonad for the maintenance of early germ cells. A mutational depletion of Cyclin B leads to a complete missing of germ cells in the adult testes and their significant loss in the ovaries. However, the requirements for Cyclin A expression for the survival of early germ cells are currently obscure. It is known that overexpression of Cyclin A or expression of its nondegradable form leads to a rapid loss of GSCs in the ovaries (Kotov, 2016).

This study found that the previously published cycB testis phenotype mimicked that in the case of bel6/neo30 mutants and germline-specific RNAi belKD. It was determined that both of the mitotic cyclins, but not Cyclin E and Cdc2, were significantly decreased in the belEY08943/neo30mutant testes. Furthermore, a considerable decrease was found of cycA and cycB mRNA levels. These results suggest a specific contribution of Belle to the transcriptional regulation of mitotic cyclins in the germline. It was also revealed that the constitutive level of Cyclin B expression in control testes was significantly lower than in control ovaries. Assuming that Belle regulates mitotic cyclins in a similar way in the germline of both sexes, it is believed that deficiency of Belle has a more severe effect on spermatogenesis, due to a sharply reduced level of Cyclin B protein below a threshold, whereas its dose in the bel6/neo30 ovaries is still sufficient to allow mitosis to occur. In support of this hypothesis a partial rescue was achieved of the RNAi belKD testis phenotype by transgenic germline-specific expression of Cyclin B, but not by Cyclin A overexpression (Kotov, 2016).

The reduction of cycB transcription in belEY08943/neo30 testes places cycB downstream of bel. In rescue experiments a third copy of cycB was added to the system employing the germinal nos-Gal4 driver in combination with UAS-bel RNAi hairpin. It is assumed that the reduction of Belle in RNAi belKD testes would negatively affect the expression of both endogenous and transgenic cycB. In accordance with this assumption only a partial restoration of the Cyclin B protein level and only partial rescue of spermatogenesis was achieved. The data indicate that at least one crucial requirement for Belle in early germ cells is relevant to Cyclin B level maintenance for ensuring germ cell mitosis (Kotov, 2016).

To date, evidences of participation of DDX3 proteins in cell cycle control both at the level of transcription and translations have been obtained. DDX3 specifically cooperates with transcription factor Sp1 to positively regulate the transcription of p21waf gene. A temperature-sensitive mutation of ddx3 gene in golden hamster cell culture at nonpermissive temperature leads to G1 arrest, which is accompanied by a decline of cycA mRNA and rather suggests the transcriptional level of regulation for cycA. It has been shown that in human HeLa cells DDX3 interacts with the GC-rich, highly structured 5'UTR of Cyclin E1 mRNA and regulates its translation initiation and a knockdown of DDX3 delays the entry to the S phase. DED1, a Schizosaccharomyces pombe homolog of DDX3, is involved in the translational control of B-type cyclin mRNAs (Cig2 and Cdc13), which have extended and expectedly highly structured 5'UTRs. It is known that only a single cyclin-dependent kinase and two B-type cyclins regulate both the S phase and mitosis in yeasts. Indeed, temperature-sensitive mutations of ded1 gene inhibit B-type cyclin translation and arrest cell cycle at both S phase and G2/M transition, whereas both cig2 and cdc13 mRNA levels remain unchanged (Kotov, 2016 and references therein).

This study has presented experimental evidence that Belle has specific and essential functions in the male germline associated with proper transcriptional regulation of mitotic cyclin expression. The testis phenotype observed in Drosophila is similar to the SCOS phenotype in human testes, indicating a conserved function of DDX3 in spermatogenesis. Understanding the molecular basis for DBY (DDX3) functions in mammalian germ cell maintenance has proven to be challenging. The functions and regulation of A-type and B-type cyclins in mammalian spermatogenesis are not clearly understood. In this case, a study in the Drosophila model provides a useful insight into the mechanism of GSC maintenance in the male germline. The current findings support a mechanism according to which the determination of the fate of male GSCs is closely connected with the control of mitosis via the regulation of mitotic cyclin levels (Kotov, 2016).

During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. This study characterized the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila. It is proposed that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes (Sechi, 2019).

Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during post-meiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex, and testis-specific TFIID (tTFIID) complex. How the action of these protein complexes is coordinated and which other factors are involved in the regulation of transcription in spermatocytes is not well understood. This study shows that the bromodomain proteins tBRD-1 and tBRD-2 regulate gene expression in primary spermatocytes and share a subset of target genes. The function of tBRD-1 was essential for the sub-cellular localization of endogenous tBRD-2 but dispensable for its protein stability. Comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depend on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22, and the tTAF Sa (Theofel, 2017).

In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. This study shows that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism (Insco, 2012).

Adult stem cells act throughout life to replenish differentiated cells lost to turnover or injury. In many adult stem cell lineages, stem cell daughters destined for differentiation first undergo a limited number of mitotic divisions to amplify cell number prior to terminal differentiation. This transit amplifying (TA) division strategy may protect large long-lived animals from tumorigenesis by minimizing the number of stem cell divisions required for tissue homeostasis and preventing accumulation of oncogenic mutations in progenitor cells due to programmed differentiation. The mechanisms that limit the number of TA divisions and initiate terminal differentiation thus may provide tumor suppressor function, and defects may contribute to progression toward cancer in adult stem cell lineages (Insco, 2012).

This study investigated the mechanisms that force TA cells to stop proliferating and initiate terminal differentiation in the Drosophila male germline adult stem cell lineage. Drosophila male germline stem cells (GSCs) reside in a niche at the tip of the testis, attached to somatic hub cells and flanked by somatic cyst stem cells (CySCs). When a GSC divides, one daughter remains in the niche and self-renews, while the other is displaced away and initiates differentiation. The resulting gonialblast, which is enveloped by a pair of CySCs, proceeds through four synchronous TA divisions with incomplete cytokinesis, producing a clone of 16 interconnected germ cells. These 16 mitotic sisters normally stop proliferating, undergo premeiotic DNA synthesis in synchrony, and switch to the spermatocyte program of cell growth, meiosis, and terminal differentiation. Because TA sister cells are contained within a common somatic cell envelope, are joined by cytoplasmic bridges, and divide in synchrony, mutations that cause overproliferation of TA cells can be easily identified (Insco, 2012).

The bag of marbles (bam) gene is required cell autonomously for TA spermatogonia to stop proliferating and enter the spermatocyte differentiation program. Male germ cells mutant for bam undergo several extra rounds of mitotic TA division, fail to differentiate, and eventually die. The number of TA divisions appears to be set by the time required for Bam protein to accumulate to a critical threshold. Bam protein is normally first detected in 4-cell cysts, increases to a peak in 8-cell cysts, and is degraded in early 16-cell cysts immediately after premeiotic DNA replication. Lowering the bam dosage slowed Bam protein accumulation and delayed the transition to differentiation, whereas early accumulation of Bam protein caused a premature switch to differentiation (Insco, 2012).

Bam, a protein with no recognizable domains, acts with a partner, benign gonial cell neoplasm (Bgcn), discovered in a genetic screen for Drosophila tumor suppressors. bam and bgcn have similar mutant phenotypes, and Bam protein directly interacts with Bgcn in Drosophila ovaries or when coexpressed in cultured cells or yeast. Bgcn is related to the DExH-box family of RNA-dependent helicases, indicating that Bgcn, and with it Bam, may regulate RNA (Insco, 2012).

Consistent with a role in translational repression, Bam protein binds the translation initiation factor eIF4A. Furthermore, expression of Bam and Bgcn in Drosophila cultured cells resulted in a 4-fold reduction in expression of a luciferase reporter coupled to the 3′ untranslated region (UTR) of e-cadherin messenger RNA (mRNA), and tethering Bam to the 3′ UTR induced translational repression of the attached reporter. In female germ cells, Bam and Bgcn allow the onset of differentiation through translational repression of nanos (nos) via the nos 3' UTR. However, direct interaction of Bam or Bgcn protein with e-cadherin or nos mRNAs has not been demonstrated (Insco, 2012).

This study identified the microRNA (miRNA) regulator and TRIM-NHL (tripartite motif and Ncl-1, HT2a, and Lin-41 domain) family member Mei-P26 both as a regulator of Bam protein accumulation and, subsequently, as a direct target of translational repression by Bam and Bgcn in male germ cells. Mei-P26 function facilitates both the switch from mitosis to meiosis and spermatocyte differentiation. In mei-P26 mutant males, Bam protein failed to accumulate to its normal peak levels. The overproliferation of TA cells in mei-P26 mutant testes was suppressed by expression of additional Bam, suggesting that the continued TA cell proliferation in mei-P26 mutant males is due to the failure of Bam protein to reach the threshold required for the switch to the spermatocyte state. In turn, Bam specifically binds the mei-P26 3' UTR, and Bam and Bgcn function are required for translational repression of mei-P26 via its 3' UTR in vivo. Mutating two potential let-7 target sites within the mei-P26 3' UTR derepressed reporter expression in vivo and disrupted Bam binding in vitro. These data suggest that a stepwise progression in regulatory states from [Mei-P26 on/Bam off] to [Mei-P26 on/Bam on] to [Bam on/Mei-P26 off], choreographed by translational regulation, accompanies the switch from TA cell proliferation to terminal differentiation in the Drosophila male GSC lineage (Insco, 2012).

It is proposed that Mei-P26 and Bam act in a regulatory cascade based on translational control to affect the switch from TA cell proliferation to spermatocyte differentiation in the Drosophila male GSC lineage. First, wild-type function of Mei-P26 in TA cells facilitates accumulation of Bam protein. Consistent with this model, a mei-P26 hypomorphic allele enhanced the overproliferation of germ cell cysts in a bam/+ heterozygote. Furthermore, the finding that adding one extra copy of bam is sufficient to rescue the early germ cell overproliferation phenotype of mei-P26 mutant males indicates that allowing normal accumulation of Bam is the major role of Mei-P26 in regulating proliferation of early male germ cells. Second, as Bam protein levels rise, Bam and Bgcn repress translation of mei-P26 via its 3' UTR in late TA cell cysts. As a result, GSCs, gonialblasts, and two-cell cysts begin with Mei-P26 expressed and Bam off and transition to 4-cell and early 8-cell cysts wherein both Mei-P26 and Bam protein are expressed. In late 8-cell and early 16-cell cysts, Bam protein levels are high, causing Mei-P26 protein to drop to very low levels. Finally, in early spermatocytes, Mei-P26 levels rise again after Bam protein disappears to facilitate normal differentiation of spermatocytes and spermatids (Insco, 2012).

Recent data suggest that Mei-P26 and related TRIM family proteins may function in the miRNA pathway. Mei-P26, two of its Drosophila homologs, and several mouse homologs have been shown to interact structurally with RISC effector proteins such as Ago-1. Mei-P26 protein localized to cytoplasmic puncta in early male and female germ cells, similar to the punctate distribution of mouse TRIM71. Many of the Mei-P26 puncta colocalized with the RISC component GW182, which accumulates in processing bodies that consist of enzymes involved in mRNA translational repression and degradation. The action of Mei-P26 in early male germ cells may facilitate accumulation of Bam protein by repressing an intermediate negative regulator of Bam. For example, Mei-P26 may function in TA cells to facilitate the accumulation of Bam protein through decreasing the function of the RNA-binding protein HOW. Previous studies suggest that HOW represses Bam expression in early male germ cells. In wild-type testes, HOW protein was expressed in early cells, including GSCs, gonialblasts, and two-cell cysts. However, in mei-P26 mutant testes, HOW protein perdured throughout the overproliferating cysts. Alternatively, given that Mei-P26 also contains a RING domain, it could facilitate degradation of an intermediate that normally degrades Bam. Bam has a C-terminal PEST sequence, a motif that targets proteins for ubiquitination and turnover by the proteasome, and expression of Bam lacking the PEST sequence resulted in early accumulation of high levels of Bam protein and a premature switch to the spermatocyte state (Insco, 2012).

As Bam protein peaks in late TA cells, it acts with its binding partner Bgcn to repress translation of mei-P26 mRNA via sequences in the mei-P26 3' UTR. Bam protein specifically binds the mei-P26 3' UTR, suggesting that Bam and Bgcn act directly as translational repressors. Translational regulation via 3' UTR sequences frequently blocks formation of the translation initiation complex by inhibiting interactions between the cap binding protein eIF4E and the 5′ cap or the rest of the eIF4F complex. Bam protein physically interacts with eIF4A independent of RNA, and eIF4A/+ partially suppressed the phenotype of bam mutants in both the male and female germline systems, raising the possibility that Bam, recruited to a target 3' UTR as part of a translational repressor complex, may block translation initiation by antagonizing eIF4A. Mutating two potential let-7 binding sites within the mei-P26 3' UTR led to derepression of the in vivo reporter and disrupted binding of Bam to the mei-P26 3' UTR. These data raise the possibility that let-7 may work with Bam and Bgcn to translationally repress Mei-P26 in TA cells. In addition, introducing the let-7-C^GK1 loss-of-function allele into a bam^Δ86/+ mutant background enhanced the bam heterozygous mutant phenotype, suggesting that let-7 and Bam may share additional targets within the testes (Insco, 2012).

Mei-P26 appears to play two distinct roles in the female germline as well: an early function in GSC maintenance and a later function required for cystocytes to switch to nurse cell and oocyte differentiation. However, there are also important differences between the male and female germline. Although Mei-P26 protein levels decreased when Bam was expressed in female germ cells, low levels of Mei-P26 were still detected. Bam and Bgcn may inhibit mei-P26 translation in female germ cells, although probably not to the same degree as in males. Notably, the mei-P26 3' UTR cloned from testes lacked the Vasa binding sites shown to be important for Mei-P26 expression in female germ cells. In addition, Bam is active at an earlier stage in the ovary, wherein the function of Bam is necessary for female GSCs to initiate the TA divisions rather than exit the TA divisions, as in males. Finally, Bam and Bgcn may have different mRNA targets in the female germline. In the female, Bam action directly or indirectly represses translation of the translational repressor Nanos, allowing the expression of proteins that initiate germ cell differentiation from the stem cell state. However, Nanos does not appear to play the same role in male as in female GSCs. Thus, the core machinery of Bam, Bgcn, and Mei-P26 probably acts through similar molecular mechanisms in female and male germ cell differentiation, but at a different point in the differentiation pathway, with different regulators and, most likely, on different targets (Insco, 2012).

Strikingly, as is shown in this study for the Drosophila male germline, the switch from mitosis to meiosis is also controlled by a regulatory network based on translational control in the C. elegans germline. BLAST and ClustalW alignments revealed that Bgcn, a core component of the switch mechanism in the Drosophila germline, is a homolog of C. elegans proteins Mog1, Mog4, and Mog5, which are required for stopping mitosis and repressing target-mRNA translation via the 3' UTR (Insco, 2012).

The requirement for TRIM-NHL proteins to facilitate the switch from proliferation to differentiation may be a widely conserved feature in many adult stem cell lineages. In Drosophila, loss of the Mei-P26 homolog dappled/wech causes large melanotic tumors, suggesting the continued proliferation of blood cells. Likewise, loss of the Drosophila Mei-P26 homolog brat in TA cells in certain neural lineages leads to brain tumors that are highly proliferative, invasive, transplantable, and lethal to the animal. In mammals, the mouse Mei-P26 homolog TRIM32 is necessary and sufficient for differentiation in neural lineages, and progenitor cells lacking TRIM32 retain proliferative status. Thus, elucidating the mechanisms by which Mei-P26 homologs and their interacting structural and regulatory partners control the switch from proliferation to differentiation in adult stem cell lineages may uncover a new class of tumor suppressors that act at the level of the developmental program rather than cell-cycle progression (Insco, 2012).

Different trans-acting factors presumably regulate the translational and transcriptional activities of the TCE. For genes specifically expressed in testes, the TCE functions as an RNA element in the 5′ untranslated region of mRNAs to repress translation and as a DNA element in the core promoter of genes to activate transcription. Testis extracts are competent to form RNA-protein and DNA-protein complexes with the TCE in vitro, but specific nucleic acid-binding factors have not been identified [30]. Data presented in this study has shed some light on the DNA-protein complex. The variable location of the TCE raises the possibility that the TCE is a short-range enhancer that is bound by a transcription activator protein rather than a core promoter element that is bound by a component of the basal transcription machinery. Indeed, the TCE is unlikely to serve as a binding site for tTFIID since all TCE-containing genes did not require tTAFs for high-level transcription. As a short-range enhancer, the TCE may function analogously to OVO binding sites that regulate the transcription of genes in the ovary. The lack of a strict requirement for tTFIID predicts the existence of a location-specific core promoter element that is recognized by tTFIID and functions in partnership with TCE at a subset of TCE-containing genes to determine testis-specific transcription. The predicted element is likely to only function in partnership with the TCE since tTAF mutations did not affect the transcription of genes that lacked a TCE. Additionally, the predicted element is likely to be of low sequence complexity since it was not identified in the bioinformatic analyses that minimally required a motif size of 5 nt. Finally, it is likely that transcription of TCE-containing genes is dependent on epigenetic features of the genome, such as nucleosome occupancy and histone modification. Thus, the TCE is a component of a combinatorial control mechanism that determines testis-specific transcription (Katzenberger, 2012).

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. This study isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, this study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes (Moon, 2011).

During spermatogenesis, the coordinated action of many gene products is essential for proper differentiation of sperm. In Drosophila, ∼25% of all genes expressed in the testis are testis-specific or testis-enriched, and most of these genes are transcribed in primary spermatocytes and stored until needed. Testis-specific gene regulation programs might ensure the coordinated expression of this large number of genes from transcription to translation. In Drosophila, two distinct classes of meiotic arrest genes, aly-class and can-class genes, represent testis-specific transcription regulation modules. The can-class genes, cannonball (can), spermatocyte arrest (sa), meiosis I arrest (mia) and no hitter (nht), encode the testis-specific TBP-associated factors (tTAFs), suggesting that their products form a testis-specific TFIID complex in primary spermatocytes. Interestingly, tTAFs, together with Polycomb group (PcG) proteins, mainly localize to a subcompartment of the nucleolus, rather than to euchromatin. Lack of tTAFs not only disrupts the nucleolar localization of PcG proteins but also causes PC to accumulate at tTAF target promoters. These findings suggested that tTAFs might also antagonize the Polycomb repressor complex (PRC1) to control the coordinated transcription of target genes (Moon, 2011).

Most aly-class gene products form tMAC, a testis-specific meiotic arrest complex paralogous to Myb-MuvB. The aly-class gene products are mainly localized to euchromatin in primary spermatocytes, and this localization is essential for their function, suggesting that the major role of the tMAC complex is transcriptional activation of testis-specific genes. Thus, it now seems evident that the coordinated transcription of testis-specific genes is regulated by two specific complexes: tTAFs and tMAC. However, little is known about post-transcriptional regulation in the testis. This study shows that the THO complex, an evolutionarily conserved complex involved in the co-transcriptional formation of export-competent messenger ribonucleoproteins (mRNPs), is a novel regulator of Drosophila spermatogenesis (Moon, 2011).

The THO complex was first found in budding yeast, Saccharomyces cerevisiae, as a multisubunit protein complex composed of Hpr1, Tho2, Mft1 and Thp2. Yeast cells that lack the THO complex show transcription impairment and transcription-dependent hyper-recombination phenotypes, implying that the THO complex connects transcription elongation to mitotic recombination. Subsequent studies showed that the THO complex, together with the mRNA export adaptor proteins Yra1 and Sub2, forms a larger complex called TREX (transcription-export complex), which is required for the co-transcriptional export of bulk mRNAs. Metazoans also have a functional homolog of the THO complex, but its subunit composition and function are slightly different from those of budding yeast. The metazoan THO complex lacks Mft1 and Thp2, but contains three other subunits, THOC5, THOC6 and THOC7, instead. Unlike budding yeast, metazoan cells require the THO complex for nuclear export of only a subset of transcripts, but almost nothing is known about the common features of the target transcripts. Although it is now clear that, at the single-cell level, a role of the THO complex in mRNP biosynthesis is conserved throughout evolution from yeast to human, its exact role in various types of cells at the multicellular organism level is still largely unknown. Recent studies using conditional knockout or a hypomorphic mutant mouse model have provided evidence that the THO complex has specific roles in cell differentiation during development. It has also recently been reported that the Drosophila THO complex is required for normal development through collaboration with E(Y)2 (or ENY2), a multifunctional protein important for transcription activation and mRNA export (Moon, 2011 and references therein).

This study reports that a novel meiotic arrest gene, garmcho (garm), encodes the Drosophila THOC5 homolog. Flies lacking THOC5 showed complete male sterility with a meiotic arrest phenotype. Interestingly, unlike any other known meiotic arrest mutants, the nucleolar structure was severely disrupted in garm mutant primary spermatocytes. Both tTAF and PC proteins were abnormally distributed, whereas the expression and nuclear export of the three tTAF target mRNAs examined were mainly unaffected in mutant primary spermatocytes. Taken together, these data provide additional evidence that the THO complex is involved in a specific cell differentiation program, Drosophila spermatogenesis, probably by participating in the establishment or maintenance of nucleolar integrity in spermatocytes (Moon, 2011).

The phenotypes of garm are different from those of other meiotic arrest mutants. First, the chromosomes were more condensed in arrested mutant spermatocytes than in wild-type spermatocytes. In other known meiotic arrest mutants, spermatocytes have chromosomes either similar to or less condensed than wild type. Second, unlike other mutants, the nucleoli were severely disrupted in garm mutant primary spermatocytes. Taken together, these findings suggest that garm represents a novel class of meiotic arrest gene, and that its gene product might play a role distinct from that of other meiotic arrest gene products, which are involved in the transcription of target genes. Indeed, THOC5 (encoded by garm) is a subunit of the THO complex, which is an evolutionarily conserved protein complex required for mRNP biosynthesis. So far, garm (thoc5) is the only meiotic arrest gene whose gene product is likely to be involved in a post-transcriptional step (Moon, 2011).

THOC5 is likely to function in spermatocytes as a subunit of the THO complex. First, all subunits of the THO complex examined were not only colocalized in spermatocytes but also co-immunoprecipitated with each other, suggesting that they form a stable complex in spermatocytes. Second, in mutant spermatocytes lacking one of the THO subunits, not only was the localization of other THO subunits disrupted, but also their expression level was significantly reduce. Third, hypomorphic mutants of both thoc6 and thoc7 appeared to show mild disruption of the nucleoli, although they did not show the typical meiotic arrest phenotype (Moon, 2011).

What is the specific role of the THO complex in Drosophila spermatogenesis? The finding that the nucleolar structure was severely disrupted in thoc5 mutants suggests that the THO complex might be required for the proper organization of the nucleolus. In accordance with this, all subunits of the THO complex accumulate at the peri-nucleolar region in pre-meiotic spermatocytes. Recent progress in understanding of the nucleolus suggests that not only does it function as the ribosome-producing factory, but it also regulates mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles. Consistent with these non-traditional functions, many proteins unrelated to ribosome assembly are found in the nucleolus. Moreover, the integration of many different sources of protein-protein interaction data showed that the spliceosomal complex is one of the major protein complexes in human nucleolus, suggesting that the spliceosomes are structural components of the nucleolus. Thus, it is speculated that the nucleolar disruption found in thoc5 spermatocytes might be caused by malformation of the spliceosomal complex, because the THO complex associates with splicesomal proteins independently of transcription. One of the common phenotypes caused by nucleolar disruption in mammalian cells is the p53-mediated cellular stress response, which includes cell-cycle arrest and apoptosis. To test whether the meiotic arrest phenotype seen in this study was also mediated by p53, genetic interactions were examined between thoc5 and p53. However, lack of p53 failed to suppress the meiotic arrest phenotype caused by thoc5 , suggesting that p53 is not required for the meiotic arrest phenotype seen in thoc5 mutant spermatocytes. This also suggested, if nucleolar disruption was the cause of the meiotic arrest, that some other unknown pathways are required for cell-cycle arrest in Drosophila spermatocyte. Alternatively, the THO complex might simply be required for the nuclear export of mRNAs for meiotic cell-cycle regulators, such as CycB or twine (Moon, 2011).

The finding that PC localization to the nucleolus requires tTAFs raised a hypothesis that the nucleolus acts as a sequestering compartment for counteracting transcriptional silencing by PcG proteins in Drosophila spermatocytes. If this is the case, transcription of tTAF target genes might be affected in thoc5 mutant spermatocytes because the nucleolar localizations of both PC and the tTAF SA are abnormal in the thoc5 mutant. Surprisingly, however, tTAF target genes were transcribed; and even more surprisingly, their transcripts were still exported to cytoplasm in thoc5 mutant spermatocytes, suggesting that the nucleolar localization of tTAFs and PcG proteins is not essential for counteracting transcriptional silencing by PcG proteins. In addition to this, a male-sterile gene, bol, which is a target of another testis-specific meiotic arrest complex, tMAC, was also expressed and exported independently of THOC5. Taken together, these results suggest that the meiotic arrest phenotype in thoc5 might not be caused by the failure of the canonical THO function, mRNA export (Moon, 2011).

The finding that don juan (dj) transcript normally appeared to be retained in the nucleus at the pre-meiotic stage of spermatogenesis is very interesting. The transcription of dj mRNA is known to be initiated in early spermatogenesis, but its translation is normally delayed until meiotic divisions are completed. Although it has been reported that translational repression of dj mRNA is mediated by the TRE (dj translational repression element) located at the 5'-UTR, it is speculated that nuclear retention of dj mRNA might be an additional mechanism by which dj mRNA is translationally suppressed. In mammalian cells, a novel regulation mechanism of gene expression through RNA nuclear retention has been recently proposed. In this model, certain mRNAs containing elements for adenosine-to-inosine editing within their 3'-UTR are retained in nuclear paraspeckles, and may be released when the demand for their protein products increases. Although it is unclear whether Drosophila has a similar regulatory mechanism, the nuclear retention of dj mRNA in pre-meiotic spermatocytes might be a sign of the existence of such a mechanism. Further studies are required to clarify this issue. From the finding that nuclear mRNA retention is not generally applicable to other tTAF target genes, the possibility that the dj probe might cross-hybridize with some other nuclear RNAs concentrated in a certain nuclear structure, such as the Y-loop, cannot be ruled out. However, this is unlikely because the signal was absent in mia mutant spermatocytes in which dj is not expressed; moreover, the nuclear dj message was still detectable in the testes of XO males, which lack the Y-loop. A previous report (Santel, 1997) that showed a strong cytoplasmic signal of dj mRNA by whole-mount in situ hybridization with colorimetric detection is also inconsistent with the current result. However, this study also failed to detect a clear signal of dj massages in the nucleus by a similar method. Thus, the discrepancy was probably due to methodological differences. Whether the signal detected by a dj anti-sense probe represents genuine dj message or not, nuclear retention of this RNA appears to be dependent on the THO complex (Moon, 2011).

The THO complex is not a testis-specific protein complex. Why, then, might the testis be more sensitive to loss of THOC5 than other tissues are? There are possible reasons for this. First, THOC5 might not be essential for the function of the THO complex. In the thoc5 mutant, a significant amount of HPR1 was still detected in the nucleus, although its level was greatly reduced. Interestingly, the residual HPR1 was mainly located near the chromatins rather than the nucleolus. Thus, the residual subunits might still have some degree of activity, and this residual activity might be sufficient for most cells, but not enough for other cells, such as spermatocytes. Second, in addition to mRNP biogenesis, the THO complex might have a non-canonical function in the spermatocytes, such as the organization and/or maintenance of nucleolar structure, and possibly the localization of some nuclear proteins and RNAs. Third, spermatogenesis requires the coordinated expression of a large number of genes. To ensure that the spermatocytes regulate gene expression in a coordinated manner from transcription to nuclear export, the THO complex might still have a role, although its specific target mRNA was not detected (Moon, 2011).

In summary, disruption of THOC5 caused severe defects in the primary spermatocyte nucleus, including nucleolar disruption, abnormal distribution of proteins (SA and PC) and an RNA transcript (dj), suggesting that it has a role in the establishment or maintenance of subnuclear structure in Drosophila primary spermatocytes. The main causative factor of meiotic arrest in thoc5 mutants might be the disruption of subnuclear structure rather than the defect in nuclear mRNA export (Moon, 2011).

In addition to the meiotic arrest phenotype reported in this study, the thoc5 mutant also has other phenotypes, including wing bubble and uncoordinated behavior. Recent work also showed that longevity and tolerance to environmental stress were significantly reduced in the THO mutants. This suggests that the role of the THO complex is not limited to spermatogenesis, but is also important for other types of cells. When this study examined whether a similar nucleolar defect is seen in other cell types, no similar defect was found in other cell types except the salivary gland cells. This suggests that the specific role of the THO complex is different depending on the cell type. It will be interesting to clarify the specific roles of the THO complex in other types of cells (Moon, 2011).

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, Prp3 was demonstrated to be essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. Two other splicing factors were identified in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. The results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes (Chen, 2019).

Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. This study reports that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. These results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis (Reina, 2018).

Centrobin was initially identified in humans through a yeast two-hybrid screen for proteins that interact with the tumor suppressor BRCA2. Like human Centrobin (referred to as CNTROB in humans), its homologues in other species are components of daughter centrioles in different cell types. In mammals, CNTROB has been reported to be required for centriole duplication and elongation as well as for microtubule nucleation and stability through its binding to tubulin and its effect in stabilizing centrosomal P4.1-associated protein (CPAP) (Reina, 2018 and references therein).

Drosophila melanogaster Centrobin is a key determinant of mother/daughter centriole functional asymmetry in larval neuroblasts and type I sensory neurons. In neuroblasts, CNB functions as a positive regulator of microtubule organizing center (MTOC) activity during interphase: its depletion impedes daughter centrioles to assemble an MTOC, whereas mother centrioles carrying ectopic CNB become active MTOCs. This activity is dependent on CNB phosphorylation by PLK1/POLO on three conserved sites and involves the function of pericentrin-like protein (PLP) and BLD10/CEP135. In Drosophila type I sensory neurons, CNB functions as a negative regulator of ciliogenesis: CNB depletion enables daughter centrioles as functional basal bodies that template ectopic cilia, and mother centrioles modified to carry CNB cannot function as basal bodies (Reina, 2018).

The function of a daughter centriole protein like CNB as a negative regulator of ciliogenesis opens the question of CNB's role in Drosophila primary spermatocytes where after centrosome duplication all four centrioles, mothers and daughters alike become basal bodies that assemble axoneme-based cilium-like structures, which are the precursors of sperm flagella. To investigate this issue, this study examined CNB localization and function in spermatogenesis (Reina, 2018).

As in other cell types in Drosophila and other species, CNB localization in spermatogonial cells was asymmetric, restricted to only one of the two centrioles that were labeled with anti-SAS4 antibodies. However, after the last (fourth) round of spermatogonial mitosis, the resulting early primary spermatocytes presented CNB equally distributed on the two centrioles of each pair (Reina, 2018).

After centrosome duplication, all four centrioles of each spermatocyte grow significantly in length, migrate to the cell surface, and become basal bodies that template a short axoneme that forms a protrusion covered by the cell membrane. Recent studies refer to this protrusion as the cilium-like region (CLR) or the ciliary cap (Reina, 2018).

To determine CNB localization in the basal body-CLR complex, CNB was immunostained together with PLP, ANA1, and UNC. PLP signal was strongest on the proximal end of the basal body. ANA1 labelled the entire length of the basal body. UNC overlapped with ANA1 except on the PLP-positive proximal end, was strongest at the transition zone, and extended further distally into the axoneme. CNB signal was equal in all basal bodies; most concentrated on the proximal end of the ANA1 domain, largely overlapping with PLP. Like PLP, a very weak CNB signal could also be detected all along the basal body. These observations reveal some remarkable differences regarding CNB localization, which in turn suggest important functional differences as far as ciliogenesis is concerned between early spermatocytes and sensory neurons in Drosophila (Reina, 2018).

To determine whether CNB has a function in the basal body-CLR complex, trans-heterozygous PBac(RB)Cnb[e00267]/Df(3L)ED4284 (henceforth referred to as Cnb mutant) spermatocytes were examined. The length of YFP-Asterless (ASL) signal (a proxy for centriole length) was significantly shorter in Cnb mutant than in WT males. Longitudinal EM sections from centriole pairs in which both mother and daughter were cut along their entire length confirmed this observation and revealed that daughter basal bodies were shorter than their mothers in Cnb mutant spermatocytes. This was not the case in WT spermatocytes, where it was found that mother and daughter basal bodies were of equal length. EM sections also revealed that 10 out of 16 mutant axonemes sectioned longitudinally were irregularly shaped and had abnormal axonemes with doublets that were aberrantly interrupted halfway through the CLR (Reina, 2018).

Ultrastructural details were revealed by transversal EM sections. Unlike basal bodies in sensory neurons that are made of doublets, basal bodies in WT spermatocytes are made of triplets. At the transition zone, the outermost tubule (C-tubule) progressively lost protofilaments, hence becoming an open longitudinal sheet that extended beyond the transition zone along the axoneme. At the point where C-tubule remnants and B-tubules intersect, short radial projections could be observed (Reina, 2018).

In addition to this basic layout of triplets, doublets, and open sheaths, about half of basal body-CLR complexes in Drosophila WT spermatocytes present a luminal singlet microtubule independently of the Z position of the cross-section. The presence of this luminal singlet is as erratic in Cnb mutant as it is in WT males and does not represent a Cnb mutant phenotype (Reina, 2018).

Eleven basal body-CLR complexes were serially sectioned from two Cnb mutant males. In all 11 samples, C-tubules were either misplaced outwards, away from the nearly straight line defined by the A/B doublet, or lost, leaving only duplets behind. These abnormalities extended distally along the axoneme, where C-tubule-derived longitudinal sheets were often lacking in Cnb mutant spermatocytes. The short radial projections observed in WT axonemes also appeared to be lacking in Cnb mutant CLRs, but this observation must be interpreted with caution because of the technical difficulties of detecting such small structures that are located in regions of electron-dense material (Reina, 2018).

These observations reveal that CNB is required in Drosophila spermatocytes for centrioles to properly position and stabilize C-tubules, grow in length up to full-sized basal bodies, and template normal axonemes. The results identify CNB as one of the very few proteins known so far to be required for C-tubule assembly, including Δ-tubulin in Paramecium tetraurelia and Chlamydomonas reinhardtii along with UNC and SAS4 in Drosophila. Notably, as in Cnb mutant spermatocytes, basal bodies are also shorter than normal in Sas4F112A and Unc mutant spermatocytes, hence suggesting that short centrioles and lacking or mispositioned C-tubules may be causally linked (Reina, 2018).

Drosophila SAS4 and its human homologue CPAP are closely functionally related to the corresponding Centrobin homologues CNB/CNTRB. Mutants in the N-terminal helical motif of the PN2-3 domain of CPAP and SAS4 cause overelongation of newly formed centriolar/ciliary microtubules, and so does CNTRB overexpression by stabilizing and driving the centriolar localization of CPAP. Moreover, the mutants in PN2-3 domain of CPAP referred to above result in biciliated cells, and so does Cnb loss of function in Drosophila sensory neurons. In Drosophila, SAS4 and CNB coimmunoprecipitate from embryo extracts. These data strongly suggest that the role of CNB on basal body length may be SAS4 dependent (Reina, 2018).

The ultrastructural phenotypes that were observed in basal body-CLR complexes in Cnb mutant spermatocytes persist through meiosis and are present in early postmeiotic cells. However, no observable defects were observable in meiosis. Cnb mutant males assembled fairly normal meiotic spindles and generated normal cysts of postmeiotic onion-stage spermatids that presented a uniform nuclear size, which is a very sensitive readout of faithful meiotic chromosome segregation. These results strongly suggest that shortened centrioles and lacking C-tubules have no major observable consequences on meiosis progression (Reina, 2018).

However, soon after meiosis, when the basal body attaches to the nucleus and the axoneme starts to grow, pushing the old CLR caudally, Cnb mutant spermatids presented CLRs that were much longer than those from WT cells. In WT cells at this stage, CNB signal was strongest on the basal body proximal to UNC and was also detectable over the perinuclear plasm, a cap over the nuclear hemisphere where the basal body is engaged (Reina, 2018).

Cnb mutant phenotypes were also conspicuous at later stages of elongation. Transversal sections through the tails of WT elongating spermatids showed two mitochondrial derivatives flanking the axoneme, which was almost completely surrounded by a double membrane. Among the sample of 32 Cnb mutant elongating spermatids that were analyzed in detail, a wide range was found of phenotypic variability. Five presented a fairly WT 9 + 2 configuration, whereas the other 27 included cells that had no axoneme or axonemes with missing or highly disarrayed duplets and in which the surrounding membrane was widely open. Among the latter, six had some duplets that carried C remnants, and 16 did not. Interestingly, not all duplet-bound open sheaths observed in Cnb mutant cells were C remnants. Four axonemes were found that presented one to three triplets with an attached open sheath. No such structures have been reported in WT cells. The presence of a fraction of Cnb mutant spermatids with axonemes that presented C remnants suggests that a certain fraction of Cnb mutant primary spermatocytes retain C-tubules (Reina, 2018).

In addition to lacking and disarranged axonemes, Cnb mutant elongating spermatids also presented ectopic nuclei at sections where only tails were present in WT cysts. Scattered nuclei have also been reported in other Drosophila mutants like Unc and Sas4. Head-to-tail attachment defects have been documented in the sperm of a rat CNTROB mutant strain (hd rats) that was male sterile (Reina, 2018).

The results reveal that CNB depletion brings about a spectrum of mutant phenotypes at different stages of spermatogenesis that includes shorter centrioles, mispositioned or absent C-tubules, lack of C-tubule derivatives, abnormal or absent axonemes, and scattered nuclei along elongating spermatid bundles. It cannot be discarded that CNB may have independent functions accounting for each of these phenotypes. However, the apparent pleiotropic effect of CNB loss may simply reflect the downstream consequences of centrioles that are shorter than normal and present C-tubule defects (Reina, 2018).

The different, and in some aspects opposite, roles of CNB in basal body structure and ciliogenesis in type I sensory neurons and spermiogenesis are remarkable. In neurons, CNB inhibits basal body function on the centriole to which it binds that in WT cells is only the daughter centriole. In primary spermatocytes, however, CNB localizes to both mother and daughter centrioles, which in these cells are equally able basal bodies, and acts as a positive regulator of ciliogenesis. One conspicuous difference between these two cell types is that basal bodies in spermatocytes contain C-tubules, whose assembly, positioning, and stability requires CNB. In vertebrates, where centrioles also have C-tubules, a recent study has found that in addition to its primary localization to daughter centrioles, CNTROB associates with mother centrioles at the onset of ciliogenesis, and CNTROB loss causes defective axonemal extension. These results suggest that the different effect that CNB depletion has on ciliogenesis in Drosophila neurons and spermatocytes may reflect the doublet versus triplet structure of the corresponding basal bodies (Reina, 2018).

The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes during spermiogenesis. Although, all the centrioles are able to organize the CLRs, this study found that the mother centriole docks first to the plasma membrane suggesting a new intrinsic functional asymmetry between the parent centrioles. The CLRs lack the Y-links that connect the axoneme doublets with the plasma membrane in conventional primary cilia. Moreover, the C-tubules, that are lacking in the axoneme of the primary cilia, persisted along the CLRs albeit modified into longitudinal blades. Remarkably, mutant flies in which the CLRs are devoid of the C-tubules or their number is reduced lack sperm axonemes or have incomplete axonemes. Therefore, the C-tubules are dispensable for the assembly of the CLRs but are essential for sperm axoneme elongation and maintenance in Drosophila (Gottardo, 2018).

Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. This study has identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective "9 + 2" structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15(KO) mutant. Together, these results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella (Wang, 2019).

The Drosophila zinc finger protein Zn72D was first identified as being involved in phagocytosis, indicating to have a role in immunity. It was then demonstrated to have a function in RNA splicing and dosage compensation. This study discovered a new function of Zn72D in male fertility. Knockdown of Zn72D in fly testes caused an extremely low egg hatch rate. Immunofluorescence staining of Zn72D knockdown testes exhibited scattered spermatid nuclei and no actin cones or individualization complexes (ICs) during spermiogenesis, whereas the early-stage germ cells and the spermatocytes were observed clearly. There were no mature sperms in the seminal vesicles of Zn72D knockdown fly testes, although a few sperms could be found close to the seminal vesicle. It was further shown that many cytoskeleton-related genes were significantly downregulated in fly testes due to Zn72D knockdown. Taken together these findings suggest that Zn72D may have an important function in spermatogenesis by sustaining the cytoskeleton-based morphogenesis and individualization thus ensuring the proper formation of sperm in D. melanogaster (Biwot, 2019).

Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. This study shows that combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes (Steinhauer, 2019).

Proper differentiation of germline cells into eggs and sperm is essential for the perpetuation of a species. Sperm cells in mammals and Drosophila melanogaster develop from germline stem cells that produce mitotic spermatogonia, which ultimately undergo meiosis and terminal spermatid differentiation. Drosophila has long been a powerful model organism for studying spermiogenesis, which begins after meiosis, when the syncytial spermatids derived from a single gonialblast (the differentiating stem cell daughter) begin morphological changes required for their differentiation, including mitochondrial differentiation, flagellar elongation, nuclear compaction and acrosome formation. In Drosophila, after meiosis is completed, mitochondria aggregate around the basal body on one side of the nucleus. Subsequently, the mitochondria fuse to form the 'Nebenkern'. As differentiation proceeds, spermatids form acrosomes, their nuclei remodel and compact, and sperm tails (flagella) elongate. During spermatid elongation, both axonemal microtubules (MTs) and the mitochondrial Nebenkern extend to form the flagellum, composed of a central axoneme flanked by major and minor mitochondrial derivatives. Drosophila sperm axonemes are structurally similar to other axonemes, containing a central MT pair ringed by nine outer MT doublets (Steinhauer, 2019).

In Drosophila, as in mammals, incomplete cytokinesis during sperm development leads to cytoplasmic sharing between sister spermatids. Following terminal differentiation, the inter-spermatid cytoplasmic bridges are dissolved and the spermatids' cytoplasmic contents are removed. This process in Drosophila, called individualization, is carried out by the actin-rich individualization complex (IC) that first forms adjacent to the needle-shaped spermatid nuclei, which by this point are clustered in the basal testis. The IC is composed of 64 actin cones, one for each spermatid nucleus of the germline cyst, and it shares similarities with actin comets found on endocytic vesicles. As individualization proceeds, the actin cones of the IC move synchronously away from the nuclei toward the apical domain of the testis, traversing the spermatid tails until they reach the end of the flagella. Each moving IC generates a growing cystic bulge that contains the extruded cytoplasmic contents of the individualizing cyst. When the IC and associated cystic bulge reach the end of the flagella, they form a waste bag (WB) full of discarded organelles, degradation of which may occur by an apoptosis-like program. As a result of individualization, each streamlined spermatozoon resides within its own plasma membrane, most of its cytoplasm has been extruded, and it no longer maintains connections to its sister spermatids. Once this process has been completed, mature sperm are coiled in preparation for release into the seminal vesicle, and WBs and abnormal sperm are eliminated within the base of the testis (Steinhauer, 2019).

Elegant prior studies have provided a detailed view of the polarized IC structure. At the leading edge of the actin cones, F-actin filaments form a meshwork under the control of the Arp2/3 actin-nucleating complex, whereas at their rear, F-actin is organized into parallel bundles by the activity of the actin-bundling proteins Quail/Villin, Chickadee/Profilin and Singed/Fascin. IC activities are thought to be segregated, with the front meshwork being responsible for cytoplasmic extrusion and the rear bundles responsible for movement along the sperm tails. During individualization, cones accumulate actin, and actin polymerization, but not myosin motor activity, is essential for IC progression (Steinhauer, 2019).

Previously identified combover (cmb), a gene encoding two major protein isoforms that are predicted to be largely intrinsically disordered and lack any known domains, as a Rho kinase substrate that acts as a planar polarity effector (PPE) to affect Drosophila wing hair (trichome) formation. During pupal development, each cell of the fly wing forms an actin-rich hair, surrounded by membrane, that points towards the distal tip of the wing. Polarized wing hair formation is governed by the non-canonical Wnt-planar cell polarity (PCP) pathway, during which core PCP proteins including Frizzled (Fz), Dishevelled (Dsh), Van Gogh (Vang), Prickle (Pk), Diego (Dgo), and Flamingo (Starry night) localize asymmetrically across proximal-distal wing cell borders. PCP-generated asymmetry leads to proximal enrichment of the PPE proteins Inturned, Fuzzy, Fritz and Multiple wing hairs (Mwh), which prevent ectopic wing hair formation. Additionally, Mwh prevents the formation of multiple prehairs, and it bundles actin to restrict hair formation to a single hair during trichome outgrowth. Although cmb mutants show no wing phenotype, Cmb overexpression causes formation of a multiple hair cell (MHC) phenotype that is enhanced by removal of one gene dose of Rok or the PPE genes, including mwh. Indeed, the two major isoforms of Cmb, PA and PB, physically interact with Mwh, and the MHC phenotype observed in mwh single mutants is partially suppressed in mwh cmb double mutants. It was therefore suggested that Cmb promotes actin-based wing hair formation under control of Rok, a function that is antagonized by Mwh (Steinhauer, 2019).

It was noticed that cmb mutants are male sterile. This study found that this is due to a requirement for cmb in the germline, in the actin-based process of spermatid individualization. Detailed histochemical and electron microscopic analyses of cmb mutant testes show that early cyst formation is normal. Furthermore, ICs form normally and start to move but do not remain properly aligned, and, although they initially recruit actin at normal levels, they fail to maintain and accumulate actin during IC progression. Despite evidence that Cmb affects actin-related processes (wing hair formation in the pupa and individualization of sperm), Cmb does not directly interact with actin in co-immunoprecipitation (CoIP) or co-sedimentation assays. Furthermore, it was shown that the role of cmb during individualization is independent of and distinct from PCP genes. Significantly, Cmb interacts with the axonemal component Radial spoke protein 3 (Rsp3/CG32392), knockdown of which causes individualization phenotypes highly similar to those of cmb mutants, suggesting that Cmb coordinates IC movement with the axoneme during biogenesis of functional sperm (Steinhauer, 2019).

In Drosophila, dynein regulates many aspects of development, including both male and female gametogenesis. Dynein-dynactin complex has been shown to play an essential role in spermatogenesis. It may anchor both centrosome and BB to the nucleus. In early G2 spermatocytes, dynein-dynactin diffuses throughout the cytoplasm. In late G2, however, dynein-dynactin accumulates at the nuclear periphery. Perinuclear dynein-dynactin may capture astral microtubules emanating from cortical centrosomes. Since dynein is a minus-end-directed microtubule motor, interaction between astral microtubules and anchored dynein-dynactin leads to the movement of centrosomes toward the nucleus, thus stable linkage between the nucleus and centrosomes is established, which is a prerequisite for fidelity of meiotic divisions. The coincident timing of ASUN's release to the cytoplasm and accumulation of dynein-dynactin on the nucleus imply that ASUN (asunder, previously known as Mat89Bb, essential regulator of spermatogenesis) may play a key role in mediating this cell cycle-dependent localization of dynein-dynactin. It has been found that mutation of sperm-associated antigen 4 (Spag4) resulted in sterility of male Drosophila. In the absence of Spag4, nuclei and centrioles or BBs dissociate from each other after meiosis. The dynein-dynactin failed to accumulate at the nuclear membrane, but instead, dynein-dynactin accumulated on the paired centrioles at the spermatocyte periphery. On round spermatids of Spag4 mutant, dynein-dynactin was not detected in the hemispherical cap as in wild types, but accumulated on the BB. This suggests that Spag4 may bring dynein-dynactin to the nuclear membrane, leading to subsequent downstream events for spermatogenesis. LIS1, another dynein accessory factor, directly binds several dynein and dynactin subunits through its C-terminal WD-repeat domain, and thus enhances dynein motor activity. Homozygous and hemizygous Lis-1 mutant males are uniformly infertile. In these sterile males, LIS-1 is required for recruiting dynein-dynactin to the nuclear surface and spindle poles of male germ cells. A pool of dynein anchored at the nuclear surface is thought to function in promoting stable interactions between the nucleus and centrosomes by mediating minus-end-directed movement of the nucleus along astral microtubules, thus LIS-1 may play an essential role in Drosophila spermatogenesis through dynein-dynactin's function (Wu, 2016).

By comparative proteomic analysis, previous work has found that Dmn protein was decreased in spermathecae and seminal receptacles of D. melanogaster females that mated with Wolbachia bacteria-infected males relative to those mated with uninfected males. As Wolbachia may modify sperm in male hosts and thus damage the male fertility, it is speculated that Dmn may have a function in male fertility. To investigate the role of Dmn in spermatogenesis, Dmn was knocked down in D. melanogaster testes by RNAi strategy. Knockdown of Dmn in male testes was found to result in dramatically decreased male fertility. By immunofluorescence technique and transmission electron microscopy (TEM), it was observed in the Dmn knockdown testes, the spermatid cell membrane frequently fused together, the immature sperm scattered around and bundles of individualization complexes (ICs) were disrupted. These phenotypes could be related to Lis-1 and Ddlc1 functions, since the expressions of Lis-1 and Ddlc1 were significantly reduced in the Dmn knockdown testes. These results suggest that Dmn may have an important function in spermiogenesis in D. melanogaster (Wu, 2016).

Wolbachia significantly modify host sperm and affect male fertility of their hosts. Previous work showed that Dmn protein was decreased in spermathecae and seminal receptacles of D. melanogaster females that mated with Wolbachia infected males relative to those mated with uninfected males. This study found that Dmn knockdown in male flies indeed severely impaired the male fertility. Wolbachia infection causes a more drastic downregulation in Dmn expression (14.2 fold) compared to nosGal4 driver (2.3 fold). Correspondingly, the male fertility is also different. Previous work have shown that Wolbachia-infected males gave only 8.78 ±1.03% of embryonic hatch rate (Zheng, 2011), while this study observed Dmn-hp knockdown males resulted in 28.97 ±4.14% of hatch rate. This suggests that the abundance of Dmn in testes is directly involved in male fertility. Furthermore, this study has shown that overexpression of Dmn in Wolbachia-infected male flies significantly rescue the fertility of both Dmn knockdown males and Wolbachia-infected males (Zheng, 2011), indicating that Dmn downregulation really plays an important role in inducing decreased male fertility in the presence of Wolbachia (Wu, 2016).

In Drosophila, spermiogenesis begins immediately after meiosis. During this stage the clonally related groups of 64 interconnected spermatids experience greatly morphological changes. One of the dramatic morphological changes is nuclear shaping and condensation. The shape of spermatid nucleus is changed from spherical to needle-like, and chromatin condenses as much as 200-fold. In the course of nuclear condensation, somatic constitutive histones are replaced by highly basic proteins such as protamine. The alteration of nuclear shape is driven by microtubules that emanate from the basal body (BB) and connect with the nuclear envelope. The perinuclear microtubules organize into parallel bundles, which are thought to provide support to the elongating nucleus. In the mean time, chromatin condensation occurs by switching from a histone-based chromatin structure in early round spermatid nuclei to a sperm-specific protein (SP, such as protamine)-based configuration in mature sperm nuclei. In Drosophila, the mammalian homologues of the protamine and transition protein have been identified as Protamine A (ProtA)/Protamine B (ProtB) and Transition protein like 94D (Tpl94D). This paper has shown that in the Dmn knockdown testes, some spermatid bundles were scattered before the nuclear shaping had completed. It has been reported that testis-specific β-tubulin has a pivotal role in the nuclear shaping in spermiogenesis. Cytoplasmic microtubules adjacent to the nuclei have been demonstrated to be important for nuclear shaping, which in turn may be required for the IC to assemble properly. This study did find that abundance of β-tubulin was significantly reduced around the nuclei in the Dmn knockdown spermatids than in the controls. This might result in the impairment of microtubule assembly. Hence less tubulin resulted from the Dmn knockdown might reduce the function of perinuclear microtubules, thus disrupt nuclear shaping. Some histone chaperones (such as Nap1) have been shown to play essential roles in nuclear shaping. Previous work also revealed that mutation of Hira, which encodes one of the histone H3.3 chaperones, significantly impaired male fertility in Drosophila. The role of Hira in spermiogenesis is not clear. However, HIRA has been demonstrated to have a critical function in sperm nuclear decondensation after fertilization by transforming a SP-based configuration to histone-based chromatin structure. Many cytoskeleton-related proteins were identified as histone chaperone partners. Therefore the transport property depending on Dmn may contribute to nuclear condensation and bundling through histone chaperone pathway and thus affect spermiogenesis. Consistently, during spermatogenesis of Wolbachia-infected male D. simulans, some nuclei were displaced from the apical cluster in some spermatid bundles. Thus, downregulation of Dmn in testes induced by Wolbachia infection, may be one reason that Wolbachia-infected males produce fewer progeny when crossed with Wolbachia-uninfected females (Wu, 2016).

The other dramatic change during Drosophila spermiogenesis is mitochondria. The mitochondria first aggregate at one side of the nucleus and then fuse and condense to form a nebenkern, which is connected with nucleus through BB. The mechanism by which mitochondria aggregate, fuse and condense to form the nebenkern near the nucleus is not well understood. This study found that there were multiple axoneme-nebenkern sets within a single cellular envelope in the Dmn knockdown testes. Two axoneme-nebenkern sets were also observed gathered together through two linked retracted minor mitochondrial derivatives. This suggests that interspermatid membrane deposition process is impaired in the Dmn mutant. Similar phenotypes were also observed in the rnRacGAP, Ddlc1 and parkin mutants. It was subsequently found that in the Dmn knockdown flies, the expression level of Ddlc1 was significantly decreased, indicating that Dmn could be involved in interspermatid membrane deposition, and knockdown of Dmn may result in the abnormal axoneme-mitochondrial assembly. Nevertheless, knockdown of Dmn did not affect the organization of axenome, implying that it is not likely to be involved in axonemal assembly. Furthermore, multiple mitochondrial derivatives were found associated with one axenome, which was previously reported in Lis-1 mutant. This study demonstrated that Lis-1 transcript was significantly reduced in the Dmn mutants. An earlier study showed that testis-specific knockout of Lis1 affected acrosomal vesicle fusion and disrupted spermiogenesis. The cytoplasmic Dynein-Dynactin complex is known to play a role in vesicular trafficking inside the cell in a variety of different cellular functions, including membrane fusion. In light of these evidences, it seems that Dynein-Dynactin-dependent vesicle transport would play an important role in the formation of cell envelope around each growing assembly of axoneme-mitochondria complex. Therefore, knockdown of Dmn may disrupt the Dynein-Dyanctin-dependent vesicle transport ability, thus damages the deposition of spermatid membrane (Wu, 2016).

At the end of spermatid morphogenesis, the mature sperm are invested with their own membranes in a process of individualization. Individualization requires formation of an individualization complex (IC). The IC is composed of 64 actin cones, one for each spermatid nucleus of the cyst. Actin filaments make a meshwork at the leading edge of the cones and are organized into parallel tight bundles at the rear of the cones. During individualization, the IC moves processively down the length of the cyst, extruding unneeded organelles and cytoplasm, and resolving intercellular bridges to encase each sperm cell in its own plasma membrane. This study showed that in the control testes, as individualization proceeded, the actin cone bundles of the IC moved synchronously away from the nuclei toward the caudal end of the cyst. However, in the Dmn knockdown testes, disorganized actin cones was observed, and some cones dissociated from the bundles and scattered around individually. It has been reported that several molecular pathways may contribute to the individualization process, including those involved in new plasma membrane deposition and microtubule cytoskeleton. Individualization during spermiogenesis is related to many processes that require deposition of new membrane, such as cytokinesis and spermatid elongation. These processes normally use vesicles to shuttle phospholipids from the Golg. Knockdown of Dmn may have resulted in the defect of vesicle trafficking ability and thus deposition of new membrane as discussed above. A number of vesicle trafficking factors are demonstrated to be required for individualization, including Shibire/Dynamin. Dynamin could play a role in actin deposition, perhaps with Ddlc1. Mutation of Ddlc1 disrupts synchronous movement of the actin cones and nuclear shaping and positioning during individualization. By qRT-PCR, significantly decrease in expression of Ddlc1 in the Dmn knockdown testes relative to the control fly testes. Additionally, overexpression of Ddlc1 significantly rescue the defects of male fertility caused by Dmn knockdown. Therefore, it is proposed that knockdown of Dmn dramatically impairs the transport ability of dynein-dynactin complex, thus damages the transportation of many important materials in cells. Recently, several evidences show that dynein-dynactin complex mediates transport of many cellular components, including mRNAs and organelles, and silencing Dmn causes significantly reduction in retrograde transportation of mitochondria. Given that during spermiogenesis, especially midway through nuclear shaping and spermatid elongation, a burst of post-meiotic transcription produces a set of mRNAs that are needed to be transported to the growing ends of the spermatid cysts, and numerous organelles, such as mitochondria and centrosomes, also need to be transported to special locations for remodeling and formation of sperm-specific organelles for fertilization and activation of development, knockdown of Dmn might affect male fertility by damaging transport ability for many important cellular contents and thus spermiogenesis (Wu, 2016).

In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, it is hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, these results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells (Karabasheva, 2019).

The endoplasmic reticulum (ER) cannot be formed by cells de novo and must be inherited during the process of cell division. While the essential roles of the ER in the biogenesis of proteins, lipids and steroid hormones, as well as calcium signaling, are well recognized, little is known about the molecular mechanisms that ensure proper partitioning of the ER to progeny cells. This knowledge is fundamental to understanding the role of the ER in cell division and stem cell biology, with important implications for proper development, tissue repair, and cancer. Specifically, recent findings indicate that asymmetric partitioning of the ER, and misfolded proteins that accumulate there, has a critical role in maintaining pluripotency in stem cells as they undergo rapid cycles of cell division. Related evidence that ER functions contribute to the proliferative capacity and drug resistance of cancer cells is under active investigation, with an objective of revealing novel therapeutic strategies for treating malignancies. The overall goal of the present study was to establish new understanding of the cellular mechanisms that guide the localization of the ER during cell division, bringing closer a full comprehension of the essential roles of the ER in normal physiology and disease (Karabasheva, 2019).

Recent data suggest that proper partitioning of the ER during cell division, or M-phase, depends on specific association of the organelle with astral microtubules (MTs) of the mitotic spindle in both symmetrically and asymmetrically dividing cells4. However, the specific factors that link the ER to astral MTs remain unknown. Identification of these factors is therefore an important next step in understanding mitotic ER partitioning. Most of the knowledge of ER-MT associations comes from non-dividing interphase cells, in which the ER is distributed throughout the cytoplasm with a distinct clustering or focus around centrosomes, the major MT organizing centers of cells. This distribution depends on MT motor-dependent movements of the ER toward both MT plus-ends and minus-ends, as well as stable attachments of the ER along MT filaments mediated by ER membrane proteins including REEPs, spastin, and CLIMP-6. Transport of the ER toward MT minus-ends is mediated by dynein motors, which are also responsible for focusing the ER around centrosomes where MT minus-ends are anchored. Conversely, MT plus-end transport is likely mediated by kinesins9, and also depends on association with growing MT tips mediated, at least in part, by ER membrane embedded STIM1 and STIM2 proteins10. Collectively these associations point to a carefully orchestrated interplay between MT plus-end and minus-end directed transport mechanisms that determine the cellular distribution of the ER (Karabasheva, 2019).

During the course of M-phase, there is a dramatic reorganization of the MT cytoskeleton, whereby the spindle apparatus forms with MT minus-ends anchored at the spindle poles by centrosomes. As this occurs, the majority of the ER becomes focused around the two centrosomes at the spindle poles and along astral MTs, and virtually none is found in the kinetochore region of the spindle where MT plus-ends reside. Thus, there appears to be a shift from the balanced MT plus- and minus-end directed ER distribution during interphase to predominantly minus-end directed localization around spindle poles during M-phase. Consistent with this conclusion, tracking of the ER with growing MT plus-ends is inhibited during cell division due to mitosis-specific phosphorylation of STIM1. This suppression of MT plus-end directed ER transport, and the totality of ER distribution toward MT minus-ends around spindle poles, suggests a predominant role for the MT-minus end motor dynein in M-phase specific ER distribution. Notably, dynein is highly localized to astral MTs and spindle poles in dividing cells and is required for the spindle pole localization of endosomes. However, despite this compelling case, a definitive role for dynein in M-phase ER distribution has never been directly established. Determining dynein's role in the dramatic redistribution of the ER to spindle poles in M-phase is therefore important to understanding the mechanisms that ensure proper ER partitioning to progeny cells (Karabasheva, 2019).

The primary goal of this study was to test the hypothesis that dynein is required for astral MT association and spindle pole focusing of the ER in dividing cells. This was accomplished by live timelapse imaging of Drosophila spermatocytes undergoing the first meiotic division of spermatogenesis in vivo. This experimental system is particularly well suited for this investigation, as Drosophila spermatocytes allow for the analysis of dividing cells in a physiologic environment and have contributed greatly to understanding of fundamental cell division mechanisms including spindle formation and cytokinesis. Moreover, Drosophila spermatocytes are large cells that exhibit clearly defined redistribution of the ER onto astral MTs early in meiosis. This study presents the surprising finding that although dynein is required for peri-centrosomal focusing of the ER during late interphase, it does not mediate the astral MT-dependent spindle pole focusing of the ER around centrosomes during M-phase. Surprisingly, the results further reveal that redistribution of the ER toward MT minus-ends in dividing cells is mediated by a mechanism of ER-MT association that is entirely specific to M-phase and does not operate during interphase. This report lays the groundwork for identification of this novel mechanism of ER-MT association that is essential for the process of ER inheritance (Karabasheva, 2019).

Proper distribution of the ER in dividing cells is critical, because it ensures that progeny cells receive necessary proportions of this essential organelle. Disruptions in this process may result in cells that are prone to accumulation of misfolded proteins and ER stress, as well as dysregulation of lipid homeostasis and calcium signaling, all of which are involved in a spectrum of diseases including cancer, neurodegeneration, and diabetes. However, the importance of mitotic ER distribution may extend beyond organelle inheritance. For example, it was recently demonstrated that the ER plays an essential role in restricting the distribution of damaged proteins in asymmetrically dividing stem cells. This mechanism, which depends on proper distribution of the ER around the mitotic spindle, may allow vital stem cells to protect themselves by asymmetrically shuttling damaged proteins to non-essential progeny cells. In addition, it has been suggested that the ER delivers highly localized calcium signals that are essential for proper function of the spindle apparatus. Thus, disruption of ER distribution around the mitotic spindle may impair the process of cell division itself with potentially devastating effects on development and tissue homeostasis. Conversely, therapeutic interventions that specifically target ER functions in cancer cells may prove to be effective treatments in neoplastic disease. Identification of the molecular mechanisms that regulate M-phase specific distribution of the ER is critical to understanding these multi-faceted roles for the organelle in dividing cells. The present investigation demonstrates the existence of a novel mechanism that mediates ER-MT association in a manner that is specific to M-phase and independent of dynein and other known MT motors (Karabasheva, 2019).

The first studies to examine ER distribution in dividing cells reported the organelle's distinct organization around the poles of the mitotic spindle, where MT minus-ends are clustered. Subsequent studies have confirmed this observation across multiple species, suggesting the existence of a unifying mechanism of ER partitioning that involves MT-dependent spindle pole association. Accordingly, involvement of a MT minus-end motor to move the ER toward spindle poles has been suggested in multiple studies, with dynein being the most likely candidate. Importantly, dynein associates with ER-rich microsomal fractions and is required for proper ER distribution in interphase cells and extracts, suggesting that the motor can associate with and transport ER membranes. Surprisingly however, the putative role for dynein in regulating ER distribution during cell division has not been directly tested. This question was addressed using meiotic Drosophila spermatocytes, which demonstrate a dramatic, near complete MT-dependent redistribution of the ER to spindle poles during cell division. The results clearly demonstrate a role for dynein in ER transport and localization in these cells, because ER distribution toward MT minus-ends and around centrosomes during late interphase was completely disrupted by dynein suppression. Surprisingly, however, dynein was dispensable for astral MT association and spindle pole focusing of the ER during meiosis. Importantly and consistent with previous reports, dynein suppression had dramatic effects on spindle formation and architecture in Drosophila spermatocytes including failure of centrosome migration to the nuclear envelope and complete dissociation of spindle poles from the focused kinetochore MT fibers. Nevertheless, despite these highly abnormal spindles, this study observed that early in meiosis the ER was still drawn onto the astral MTs and towards the centrosomes, despite the centrosomes' mislocalization at the cell cortex. Moreover, this redistribution of the ER occurred with the same kinetics and to the same extent as in control cells, suggesting that the mechanism of ER-astral MT association is completely independent of dynein. In this regard, data indicating that dynein association with membranous organelles is inhibited during M-phase of cell division suggests the existence of a dynamic mechanism that reciprocally regulates dynein association of the ER with MTs according to the stage of the cell cycle (Karabasheva, 2019).

An important outcome of dynein suppression in spermatocytes is that it allowed a clear separation of interphase versus M-phase mechanisms of ER association with centrosomal MTs, wherein the ER was completely excluded from centrosomal MTs during late interphase but suddenly moved along MTs toward the centrosomes early in M-phase. This reveals that an M-phase specific mechanism of ER-MT association that concentrates the ER around centrosomes is triggered at the onset of cell division, possibly due to specific cyclin/cyclin-dependent kinase activity. This conclusion is further supported by the observations that several of the mechanisms for ER-MT association that operate during interphase are in fact inhibited during M-phase, including those mediated by STIM1, CLIMP-63, and possibly dynein. Collectively, these findings suggest an enticing mechanism whereby most or all interphase mechanisms of ER-MT association are inhibited during M-phase, allowing an M-phase specific mechanism to predominate and ensure ER association with astral MTs and proper partitioning to daughter cells (Karabasheva, 2019).

The challenge moving forward is to identify the M-phase specific mechanism that associates the ER with astral MTs. Importantly, in dynein suppressed spermatocytes it was clear that the ER moved along astral MTs toward the minus-ends, suggesting that a MT minus-end motor distinct from dynein may be involved. However, the present findings indicate that neither ncd, the only other known bona fide MT minus-end motor in Drosophila, nor Klp61F, which generates pole-ward forces in the spindle, are involved in the unique M-phase distribution of ER with astral MTs. Thus, it is possible that an unidentified MT minus-end motor is required, or that a non-motor factor stably attaches ER membranes along MT fibers and tubulin flux then moves these membranes toward centrosomes. In this regard, it was recently demonstrated that human REEP3 and 4, members of the REEP1-4 family of ER membrane proteins, directly associate with MTs and play a role in spindle pole focusing of the ER37. However, whether REEP3/4 associate the ER specifically with astral MTs has yet to be determined. Drosophila have a single orthologue to human REEPs1-4, known as REEPA, and preliminary observations indicate that REEPA also is not required for astral MT association of the ER in meiotic Drosophila spermatocytes. It is also important to note that different mechanisms of ER-MT association may operate during cell division in different cell types. Thus, while the current results cannot rule out a role for dynein or other MT minus-end motors in ER partitioning in cells other than Drosophila spermatocytes, the findings do indicate that these factors are not universally required (Karabasheva, 2019).

In addition to identifying the molecular factors that link the ER to astral MTs during cell division, another important question is how does this association discriminate astral MTs from other MTs of the spindle apparatus? Certainly, suppression of interphase mechanisms plays a role, as expression of nonphosphorylatable STIM1, which remains associated with MTs during mitosis, results in mislocalization of the ER to kinetochore MTs. It is also possible that differences in tubulin post-translational modifications facilitate discrimination between different MT populations within the spindle, as demonstrated for kinesin-7 motors that carry chromosomes toward the spindle equator along detyrosinated MTs of the inner spindle (Karabasheva, 2019).

In conclusion, this study has demonstrated that an M-phase specific mechanism associates the ER with astral MTs and partitions the organelle to spindle poles in dividing cells. Surprisingly, and in contrast to many previous suggestions, this ER localization is not mediated by dynein or other known MT minus-end motors. Identification of the mechanisms involved is an important next step in better understanding the role of the ER in cell division, stem cell longevity and pluripotency, and tissue architecture. This knowledge will facilitate development of novel therapeutics for pathological processes underlying cancer and age-related tissue degeneration (Karabasheva, 2019).

The Drosophila melanogaster ATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. Although loss of Drosophila ATPCL reduces levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. This study describes that depletion of ATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for ATPCL during spermatogenesis. ATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that ATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, these results unveil for the first time an involvement for ATPCL in the regulation of meiotic cell division, which is likely conserved in human cells (Di Giorgio, 2020).

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, this study identified a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage (Galletta, 2020).

An essential element of functional flagellated sperm is proper attachment between the head, which contains the genetic material, and the tail, which generates the force for swimming. A failure in this connection can result in decapitated, decaudated, or malformed sperm, ultimately leading to reduced fertility. The most well documented human study investigated 10 infertile males with acephalic sperm. These patients showed a variety of 'abnormal head-neck configurations,' including breaks between the head and tail and sperm with nuclei laterally attached to the midpiece near the centrioles. The head to tail linkage is centered around the centriole, referred to in this context as a basal body (the term centriole is used for simplicity). The 'distal end' of the centriole templates and anchors the axoneme, the core structural element of the tail, while the 'proximal end' of the centriole forms a connection with the nuclear surface. Thus, the proximal and distal ends of centrioles play distinct and critical roles in sperm assembly. Note that the proximal end and distal end of an individual centriole are being discussed; these are distinct from the 'proximal centriole' and 'distal centriole' terms used in mammalian systems to describe the two centrioles within each sperm. Except when specifically discussing mammalian spermiogenesis, the latter terms will not be used or referenced in this study (Galletta, 2020).

A prerequisite for a tight connection between the head and tail of the sperm is the relocation of the centriole to the nuclear envelope during early spermiogenesis in Drosophila and in mammals. In Drosophila, after the exit from meiosis II, the centriole is repositioned against the reformed nuclear envelope and eventually becomes embedded in the nuclear envelope and is surrounded by an electron-dense material suggested to provide a tight connection. Similarly in mammals, the 'proximal centriole'" moves and attaches to the nucleus where an electron-dense material accumulates and the 'connecting piece' assembles around the centriole pair. In both systems, the centriole templating the flagellar axoneme has its proximal end closest to the nucleus and is positioned perpendicular to the nuclear surface (Galletta, 2020).

In mammalian model systems, there are examples of mutations that cause the head-tail connection to fail, including mutations in centriole proteins Centrin 1 and Centrobin, but little is understood at the molecular level of how these centriole proteins are involved in establishing the head-tail connection. Mutant analysis in Drosophila has identified additional players such as Asunder, Lis-1, Spag4, Yuri gagarin, and dynein and dynactin components, indicating that positioning the microtubule (MT) motor dynein at the nuclear envelope is critical. Finally, mutations in gamma tubulin ring complex proteins, which are required for proper MT formation, result in defective centriole-nuclear attachment in older developing spermatids (STs). It is believed these studies in totality suggest a model whereby dynein on the nuclear surface binds and acts on centriolar MTs to reposition the centriole to the nucleus. However, this process has never been documented in live STs, the role of the centriole itself in this process has not been examined, and the molecular mechanism that ensures correct centriole orientation and docking has not been investigated in detail (Galletta, 2020).

The highly stereotypical proximal end-on docking of centrioles to the nucleus suggests that the centriole proximal end is specialized. Many studies have carefully defined proteins uniquely positioned along the proximal-distal centriole axis. This polarized localization can convey local functions. For example, proteins that regulate centriole length such as Klp10A, Cep97, and Cp110 are positioned at the distal end of the centriole where centriole elongation is thought to exclusively occur. When a new centriole forms, proteins such as Ana2/STIL and Sas6, structural elements required for earliest steps daughter centriole formation, accumulate at the proximal end of the mother centriole. Additionally, pericentriolar material (PCM), which is critical for nucleating and organizing MTs, appears to be restricted to the centriole proximal end in mammalian systems and the meiotic centrioles of Drosophila spermatocytes (SC). This suggests that MTs are predominantly produced and anchored at the proximal end of centrioles. However, unlike distal-end protein components, the importance of restricting PCM to the proximal end and the mechanism by which this restriction is achieved are unknown. This study sought to identify the mechanism by which PCM is proximally restricted, and it was hoped, in turn, to gain insight into proximal end nuclear docking during spermatogenesis (Galletta, 2020).

Functional sperm require a stable linkage between sperm head and tail, which is mediated by the centriole. To date, little is known about the molecular mechanisms underlying defective head-tail attachment, except for a few reports identifying mutations in genes such as Spata6, Sun5, BRDT, PMFBP1, and TSGA10. Directly relevant to this study on the role of the centriole in head-tail attachment, is TSGA10 (Cep135 paralog) and two other centriole proteins -- Centrin1 and Centrobin. Thus, the limited patient sequence analysis and mammalian model system mutants point to a critical role for the centriole in head-tail attachment (Galletta, 2020).

Live imaging of centrioles in Drosophila STs revealed that the docking of the centriole to the nucleus is a two-step process. The first is 'Nuclear Search,' where the centriole searches for its docking partner, the nucleus, immediately following meiotic exit. The second is 'Nuclear Attachment,' where a stable connection of the centriole to the nuclear surface is formed. Work in Drosophila has also identified a number of mutations that ultimately result in decapitation, several of which affect the ability of the MT motor dynein to localize to the NE, or to generate pulling forces on MTs. Interestingly, studies from mice have also implicated the dynein adapter Hook1 and the sun protein SPAG4 in ensuring proper head-tail linkage. These proteins have been shown across species to position the centrosomes adjacent to the nucleus in interphase of normally cycling cells. Thus, a simple model emerges where in developing STs a conserved dynein-based system on the surface of the nucleus interacts with MTs emanating specifically from the proximal end of the centriole to draw the proximal centriole end specifically to the nuclear surface. Imaging of MTs in Drosophila suggests that this model is quite plausible, showing MTs specifically emerge from the proximal end of the centriole, which was then shown is key to proper centriole-nuclear docking. One exciting future direction is to reexamine the known Drosophila decapitation mutants to separate players that affect Nuclear Search from those that affect only the later step of Nuclear Attachment (Galletta, 2020).

While this study focuses on the formation of functional sperm, it also provides insight into centrosome architecture and how this architecture relates to function, in this case nuclear docking. In recent years, enormous progress has been made in identifying centrosome proteins and mapping their localization with nanometer precision. The challenge now is to link protein position with function; progress on this front has been most notably made at the centriole. For example Ana2/STIL, Sas6, and Cep135 form a cartwheel structure inside a new centriole at its proximal end, serving as a template for centriole symmetry. Another protein complex, Cep97, CP110, and Klp10A, localizes to the distal end and to control centriole length. Thus, subcentriolar localization and function are intimately linked. Despite PCM being documented at the proximal end of the centriole, a link between the position and function of the PCM at the proximal end has not been established. One possible role for the PCM at the proximal end is to dictate the position of daughter centriole formation. Previous work has shown that new daughter centriole nucleation requires PCM, and overexpression of PCM results in additional daughter centriole formation. However, the position of the ectopic centrioles along the proximal-distal axis was not examined, and the importance of the proximal position could not be inferred. This study provides a clear demonstration of a proximal-specific function for PCM (Galletta, 2020).

Through a series of wild-type, mutant, and misexpression experiments, this study showed that the bridge protein PLP is critical in proper centriole docking in STs. By examining plp mutant testes, it was found that PLP is a major driver of PCM recruitment or retainment at centrioles in round spermatids (RSTs). In the absence of PLP, PCM is disorganized and the centriole is improperly positioned away from the nucleus. This study then investigated how PLP itself is restricted to the proximal end. Using endogenously tagged PLP protein, single-cell RNA-seq and whole mount in situ, this study showed that proximal restriction of PLP is achieved through a reduction in PLP mRNA and protein concentration prior to centriole elongation. It will be important in future studies to determine precisely how PLP concentration is reduced; there is likely a delicate balance between decreased PLP translation, increased PLP degradation, and simple dilution of PLP as spermatocyte size increases (Galletta, 2020).

While numerous studies have shown that PLP is necessary for PCM organization around centrioles and that PLP can interact with PCM components, the precise mechanism of how PLP acts to recruit or anchor γ-tub is not known. In addition to interacting with PCM components, both PLP, and its mammalian ortholog pericentrin, have been found in complexes with γ-tub itself. It remains to be determined precisely how the multiple possible pathways through which PLP can affect the PCM function at the centriole in different contexts. Whether direct or indirect, this study shows that mislocalizing PLP on the centriole is sufficient to dictate PCM position on the centriole. When PCM is present at a more distal position along the centriole in STs, MTs emanate from the entire centriole, which can result in lateral capture to the nucleus. This lateral capture appears unstable and firm Nuclear Attachment does not occur. It is proposed that these failed, or defective, centriole-nuclear attachments do not survive the forces applied as a result of axoneme and mitochondrial derivative elongation, nuclear clustering, and/or ST individualization, ultimately resulting in sperm decapitation. This is consistent with several studies linking a failure in individualization to a failure in centriole-nuclear attachment. Additional studies will be required to determine how the tight attachment between the nucleus and the centriole forms, precisely when the Nuclear Attachment fails in STs with laterally docked centrioles and how this connection relates to the machinery that drives the massive cellular reorganization required to build sperm (Galletta, 2020).

The transcriptional mechanism to restrict protein localization this study identified is one way to achieve sub-centriolar protein compartments. Other mechanisms include specific docking-site recognition, such as the LID (longitudinal tubulin-tubulin interaction domain) domain of Sas4 recognizing the plus end of MTs at the centriole distal end and protein symmetry-breaking and coalescence as seen with Plk4's ability to concentrate into a single spot on the surface of the centriole. While it is unknown if other proteins use a transcriptional mechanism like PLP for centriole position control, single-cell RNA-seq data could help identified such proteins (Galletta, 2020).

One exciting finding from this work is that simply altering the timing of PLP expression can have major deleterious effects. This can be analogous to many human diseases that are frequently reported to correlate with higher levels of protein expression; understanding the underlying cell biology and physiology of the protein overexpression becomes quite critical. For example, overexpression of the master regulator of centriole duplication Plk4 is sufficient to promote tumorigenesis and renal cysts. Another example is seen in the case of having an extra copy of pericentrin (the ortholog of PLP), which in humans is present on chromosome 21. An increase in pericentrin protein levels by 50% can cause defects in ciliogenesis and cilia function. Therefore, understanding the role of the centriole in human disorders will not only require understanding the consequences of loss of protein function but also the consequence of protein misexpression and misregulation (Galletta, 2020)

Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalized primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. This study describes phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalized from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells (Hodge, 2021).

After centrosome duplication, centrioles elongate before the M phase. To identify genes required for this process and understand the regulatory mechanism, this study investigated the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles. An essential microtubule polymerisation factor, Orbit/CLASP, was identified that accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically for determining centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. Loss of centriole integrity was observed, including fragmentation of centrioles and earlier separation of the centriole pairs in Cp110 null mutant cells either overexpressing Orbit or harbouring Klp10A depletion. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they are unpaired. These findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells (Shoda, 2021).

The centrosome plays an indispensable role as the major microtubule-organising centre (MTOC) in a cell. During mitosis, centrosomes duplicated in the S phase move apart from each other and reach the opposite poles of the cell. Each centrosome is involved in the assembly of spindle poles, which enables construction of the bipolar spindle microtubule structure. A centrosome consists of two components: a pair of centrioles and the surrounding pericentriolar matrix (PCM). After replication of the centrioles, the longer centriole (mother centriole) engages with the shorter one (daughter procentriole) in a V-shape. When a cell enlarges in the G2 phase, the short daughter procentriole undergoes elongation to a certain length before the subsequent M phase. A single centriole consists of a microtubule doublet or triplet, which is equivalent to the cytoplasmic microtubule. Several factors localised on centrioles have been shown to be involved in the centriole elongation process. The most critical step in centrosome duplication is the duplication of centrioles, which requires stringent regulation. However, the entire mechanism underlying regulation of centriole elongation and the regulatory factors required for the process are not known. Among the centriole-associated proteins, those belonging to the kinesin-13 family are known to act as microtubule-severing kinesins. Klp10A, a Drosophila member of the family, has been shown to play an indispensable role in the regulation of centriole elongation. Based on these observations, it is speculated that some of the factors regulating microtubule length might overlap with those required for centriole elongation. It is possible that the production of centrioles of specific length requires a balance between polymerisation and depolymerisation of the triplet microtubules. However, the main factor(s) counteracting Klp10A and promoting centriole elongation remain to be identified (Shoda, 2021).

Another characteristic complex containing Cp110 is localised at the distal tip of the centriole, where it regulates the accessibility of the distal end to the shrinking and hypothetical lengthening factors, thereby regulating centriole elongation at this end. In the absence of Klp10A, the longer centrioles harbour incomplete ninefold symmetry at their ends in Drosophila cultured cells and tend to undergo fragmentation. Importantly, Cp110 depletion differentially affects centriole elongation in a species- and/or cell type-specific manner. In Drosophila S2 cultured cells, Cp110 depletion results in centriole length diminution. This effect is rescued by simultaneous depletion of Klp10A. In contrast, CP110 (also known as CCP110) depletion results in centriole elongation in mammalian cells. The centriolar microtubules were dramatically elongated in somatic cells, such as wing discs and larval brain cells, in the Drosophila Cp110-null mutant, whereas subtle elongation of the structure was observed in the premeiotic spermatocytes of the mutant (Shoda, 2021).

The premeiotic spermatocyte in Drosophila is a good model for investigating centrosomes and centrioles. Drosophila spermatogenesis involves four mitotic and two meiotic cycles for the formation of haploid spermatids. In the same spermatocyte cyst, each of the 16 cells undergoes synchronous cell growth, which can be divided into the S1 stage, corresponding to S phase, and five subsequent stages, S2 to S6, before initiation of meiosis I. The centrioles, in particular, can be studied more easily in this cell type, since these organelles dramatically elongate until the onset of meiosis and the centriole cylinder is composed of microtubule triplets. In early spermatocytes that possess a pair of centrioles initially, centrioles duplicate at S1 stage. As primary spermatocytes enter in the growth phase, centrioles migrate toward the surface where they assemble the primary cilium at the distal end of basal body. At the beginning of meiotic division I, centrioles move close to the nucleus with their associated 'membrane pocket' on the distal end of the cilium-like region (CLR). Between the CLR and the basal body there is the transition zone (TZ), which plays an important role in elongating the primary cilium of the spermatocyte. Centrioles are no longer duplicated between the two meiotic divisions. Primary spermatocytes hold two pairs of centrioles composed of nine triplet microtubules and engaged by a cartwheel structure at the proximal ends. The centriole pair is disengaged during prophase II, and, consequently, singlet centrioles organise the centrosomes of secondary spermatocytes (Shoda, 2021).

Previous studies have shown that Orbit (the Drosophila CLASP orthologue, encoded by chb) is essential for microtubule polymerisation, as it adds tubulin dimers to the plus end of the microtubules; however, its role in centriole elongation has not been examined. Hence, this study aimed to investigate whether Orbit was involved in centriole elongation in the mature premeiotic spermatocytes before male meiosis. As Orbit antagonises Klp10A, a severing factor determining the length of spindle microtubules in cultured Drosophila cells, whether Orbit was also involved in centriole length regulation was assessed (Shoda, 2021).

In addition, this study highlighted the importance of these regulators of centriole dynamics and the distal end capping proteins in the centriole elongation process using Drosophila spermatocytes. The importance of regulating the elongation of duplicated centrioles to a certain length for proper chromosome inheritance during male meiotic divisions is also discussed (Shoda, 2021).

Centrioles in Drosophila spermatocytes consist of ninefold triplets of microtubules. This study showed that the centrioles elongated to a certain length as the cells grew before male meiosis. Klp10A, which is a Drosophila kinesin-13 orthologue essential for shortening the microtubules, plays an indispensable role in regulation of centriole length. Microtubule length can be determined by the balance between polymerisation and depolymerisation of tubulin heterodimers and protofilaments. Thus, dynamic factors that promote microtubule elongation might also play critical roles in the determination of centriole length. This study has presented evidence suggesting that overexpression of Orbit results in excessively long centrioles in premeiotic spermatocytes. Conversely, shorter centrioles were observed in hypomorphic orbit mutants. These results are consistent with the idea that Orbit is essential for promotion of centriole elongation in spermatocytes. Orbit was initially identified as a microtubule-associated protein that adds tubulin heterodimers at the plus end of microtubules. Hence, the probability that Orbit might also elongate the triplet microtubules of centrioles by adding tubulin dimers, similar to its function in microtubule elongation, is high. In contrast, the kinesin-13 orthologue Klp10A acts as a microtubule depolymerising factor at the plus ends of microtubules in interphase and is an important regulator of centriole elongation. In Drosophila S2 cells, Klp10A antagonises Orbit in bipolar spindle formation and its maintenance. This study has shown that simultaneous overexpression of Orbit and depletion of Klp10A further enhanced centriole elongation. Based on these findings, it is proposed that these two factors act antagonistically to produce centrioles of specific length. Very long GFP-Orbit signals were observed that extended from basal bodies in spermatocytes overexpressing the protein. Accordingly, it is hypothesised that these are overly long axoneme microtubules produced as a consequence of excessively stimulated polymerisation of tubulins by the overexpression. Orbit, like other CLASP proteins, has a microtubule-binding activity to stimulate tubulin polymerisation at the plus end. Alternatively, a possibility cannot be excluded that Orbit polymerises itself to construct microtubule-like structures on the distal tip of the basal body. It would be interesting to investigate whether basal body and axonemal microtubules overly elongate in cells overexpressing Orbit without a fluorescent tag (Shoda, 2021).

In addition to these two factors regulating centriole microtubules, it is hypothesised that Cp110 plays a role as a cap to restrict these factors acting on the distal ends of the microtubules at an earlier stage. After Cp110 releases from the ends at mid-stage, Orbit can access the distal ends more easily and stimulate the centriole microtubules to extend to a certain length. Although the loss of the cap protein resulted in only a subtle extension of centrioles, Orbit overexpression in the absence of Cp110 can change the centriole microtubule dynamics significantly. Consequently, remarkably long centriole microtubules would be produced in the spermatocytes. Consistent with this hypothesis, Cp110Δ1 mutation also significantly enhanced the overly long centriole phenotype in Klp10A-depleted spermatocytes. By contrast, the Cp110-null mutation did not enhance the shorter centriole phenotype caused by Klp10A overexpression. Further experiments need to be performed to verify the model. The fact that Cp110 influences centriole length depending on the cell type and cellular context has been demonstrated in previous studies. It is likely that differential regulation of the conserved core components underlies ciliary basal diversity in different cell types. As argued previously, cellular-specific and tissue-specific regulation in centriole duplication may be indispensable to regenerate diverse centriole structures (Shoda, 2021).

Previous reports have not investigated whether Orbit and Klp10A are localised on centrioles or around the PCM in Drosophila mitotic cells; however, a considerable amount of Orbit accumulates in centrosomes in early embryos, Drosophila cultured cells and germline stem cells during Drosophila oogenesis and spermatogenesis. Whether the protein is localised in the PCM or in the centrioles is not clear. Similarly, studies on the cellular localisation of Orbit/CLASP orthologues in other species have also shown centrosome localisation of these proteins. Anti-human CLASP1 immunostaining in Hela cells has demonstrated that the protein is localised on centrosomes during M phase (Maiato et al., 2003). Furthermore, a CLASP orthologue in Caenorhabditis elegans has been observed in the centrosomes of its embryonic cells. These reports did not mention whether the orthologues were associated with the centrioles. Recently, it has been reported that Klp10A is dominantly localised in the TZ of the ciliary structures in spermatocytes, spermatids and sensory neurons (Persico, 2019). Centrosomal localisation of the protein has also been reported in mitotic cells and germline stem cells in both sexes. Whether Klp10A localises on the cylindrical microtubules of the centrioles or in the PCM of mitotic cells has not been demonstrated. Therefore, whether these antagonistic regulators are localised on centrioles in mitotic cells should be determined at a higher resolution. Furthermore, whether these two regulators are required for centriole length determination in somatic cells warrants further investigation (Shoda, 2021).

In addition to excessively elongated centrioles, this study observed several abnormalities in centriole organisation and structure in spermatocytes overexpressing Orbit and/or harbouring Klp10A depletion. In the absence of Cp110, the loss-of-centriole-integrity phenotypes were also enhanced. Small pieces of centrioles observed may be broken pieces of over-elongated centrioles, as observed in cancer cells. Alternatively, they may have been unpaired centrioles separated precociously from centriole pairs containing daughter procentrioles, which are smaller than normal centrioles. By contrast, the loss-of-centriole-integrity phenotypes were not observed in cells overexpressing a shortening factor, Klp10A. Hence, it is considered that improvised construction of the basal body microtubules may be associated with the loss-of-integrity phenotype; thereby, centriole engagement would be lost. Centriole fragments were found with reduced diameter in the centriole microtubules. The presence of disintegrated centrioles supports this idea, but further investigations are necessary to test the current hypothesis (Shoda, 2021).

Spermatocytes homozygous for loss-of-function mutation of Sas6 and of Ana2 commonly demonstrate premature centriole separation before meiosis. Hence, Sas6 and Ana2 are considered to be required for centriole engagement and/or maintenance of the pairs. Orbit overexpression and/or Klp10A depletion may affect centriole engagement through interfering with Sas6 (or Ana2) function. It is also possible that the premature disengagement can occur independently of Sas6 or Ana2. In addition, it has been reported that APC/C activation and activation of separate, thereby unexpected, cleavage of Scc1 (also known as RAD21) cohesin can take place in mammalian cultured cells. The possibility cannot be excluded that alteration of microtubule dynamics in centrioles by altered expression of Orbit and/or Klp10A led to unexpected APC/C activation. This hypothesis will be tested by several experiments in future work (Shoda, 2021).

Previous studies have also mentioned that Cp110-null mutant spermatocytes or syncytial-stage embryos do not show detectable defects in centrosome behaviour, spindle formation or chromosome segregation. By contrast, this study has show that disintegrated centrioles and multipolar spindle microtubules emanating from the centriolar fragments existed in Cp110-null mutant spermatocytes and cells overexpressing Orbit, as well as in Klp10A-depleted cells less frequently. Cells homozygous for the loss-of-function Klp10A mutation also display multipolar spindle structures. However, it cannot be excluded that the spindle phenotype would result from abnormal microtubule organisation caused by Klp10A mutation, rather than centriole disintegration. Surprisingly, in Cp110 mutants overexpressing Orbit and undergoing meiosis I, it was observed that unpaired single centrioles, even a part of them, could act as the MTOC. Centrosomes must be 'licensed' to function as an MTOC that nucleates microtubules, although the mechanism whereby the 'license' is granted remains unclear. Nevertheless, a recent study has reported that excessive elongation of centrioles in cancer cells is related to the generation of overduplicated, fragmented or hyperactive centrosomes that nucleate considerably more microtubules during cell division. Chromosome segregation is disturbed in cells harbouring these abnormal centrioles. Interestingly, generation of cells harbouring extra centrosomes has been suggested to be able to drive spontaneous tumorigenesis in mice. Additional studies have reported that excessively elongated centrioles in Drosophila spermatocytes affect spermatogenesis via the production of defective flagella. Consistently, immotile sperm production and significant decrease in male fertility were observed in the Cp110-mutant males with spermatocyte-specific Klp10A depletion. These observations suggested that production of excessively elongated centrioles can affect cell division and subsequent spermatogenesis. Once an abnormal spindle microtubule structure is constructed, the extra MTOC results in chromosome mis-segregation and eventually chromosome instability, as observed in cancer cells (Shoda, 2021).

Certain abnormalities such as mis-segregation of chromosomes and the resulting aneuploidy might appear in the subsequent division of cells containing abnormal centrioles. Conversely, excessively short centrioles may be inadequate as templates for the duplication of centrioles in S phase. Hence, regulation of centrosome length via the antagonistic activities of elongation and shrinking factors, such as Orbit and Klp10A, is important. However, the loss of centriole integrity and the resulting aneuploidy may occur in meiotic cells, but not in mitotic cells, which have stricter microtubule assembly checkpoints. Further investigations are required to understand how centrosome length is regulated in mitotic cells (Shoda, 2021).

Current findings suggest the presence of mutually antagonistic regulation to determine centriole length and the significance of the production of centrioles with a certain length for centriole integrity, and for assurance of proper chromosome segregation. It is believed that these findings may enable the identification of a mechanism whereby the loss of centriole integrity causes chromosome mis-segregation in cancer cells (Shoda, 2021).

Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubule-organizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. This study describes the existence of a novel, tissue-specific γ-TuRC in Drosophila. Three genes (CG7716 (t-Grip84), CG18109 (t-Grip91), and CG32232 (t-Grip128) were characterized encoding testis-specific components of γ-TuRC (t-γ-TuRC) testis-specific Grip paralogs. Presence of t-γ-TuRC is essential to male fertility. The diverse subcellular distribution was shown of the t-γ-TuRC proteins during post-meiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. The physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1 were also proven. These results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC (Alzyoud, 2021).

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. This study reports the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly (Lage, 2021).

Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. This study described and examined the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, it was demonstrated that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules, required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis (Bauerly, 2022).

Galletta, B. J., Varadarajan, R., Fagerstrom, C. J., Yang, B., Haase, K. P., McJunkin, K. and Rusan, N. M. (2023). The E3 ligase Poe promotes Pericentrin degradation. Mol Biol Cell 34(9): br15. PubMed ID: 37342879

Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in Drosophila. Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. A previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. It was hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. This study shows that PLP is subject to ubiquitin-mediated degradation and identified multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which was shown to bind to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, this study identified a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids (Galletta, 2023).

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling (Amoyel, 2016b).

This study shows that PI3K/Tor activity is required for the differentiation of somatic stem cells in the Drosophila testis. Additionally, a 'differentiation' niche was identified immediately adjacent to the stem cell niche that, through the local production of Dilps, leads to the upregulation of PI3K/Tor activity in early CySC daughters and to their commitment to differentiation. The secretion of ImpL2 by CySCs antagonizes the initiation of differentiation in CySCs by blocking available Dilps in the stem cell niche. As a result, CySCs receive little free Dilp ligands. However, as their daughters move away from the hub, they encounter increasing levels of Dilps and decreasing levels of ImpL2, which leads to the upregulation of PI3K/Tor signaling and proper somatic cell differentiation. The fact that ImpL2 is upregulated by the main self-renewal signal (i.e., JAK/STAT) in CySCs leads to a model accounting for the spatial separation of the stem cell niche and the differentiation niche (Amoyel, 2016b).

The results are consistent with a model in which autocrine or paracrine production of Dilp6by early cyst cells serves as a differentiation niche in the testis, defining where in the tissue upregulation PI3K/Tor signaling - a prerequisite for differentiation - occurs. This differentiation niche is critical for somatic development because stem cell markers like Zfh1 are maintained in the absence of signals like PI3K/Tor. Notably, JAK/STAT activity is not expanded outside of the niche upon somatic loss of PI3K/Tor signaling, suggesting that differentiation signals play a critical role in downregulating stem cell factors. Intriguingly, recent studies in the Drosophila ovary have identified a differentiation niche in this tissue: autocrine Wnt ligands produced by somatic support escort cells regulate escort cell function, proliferation and viability. Taken together, these studies reveal that at least in Drosophila gonads, there is a defined region immediate adjacent to the stem cell niche where autocrine production of secreted factors induces the differentiation of somatic cells, which in turn promote development of the germ line (Amoyel, 2016b).

Several studies have examined the role of insulin signaling in gonadal stem cells. In both testes and ovaries, systemic Dilps have been shown to affect stem cell behavior. In both tissues, nutrition through regulation of systemic insulin controls the proliferation rate of GSCs. The current data showing that Akt1, Dp110 or Tor mutant CySC clones proliferate poorly are consistent with these findings and indicate that basal levels of insulin signaling are required for the proliferation and/or survival of both stem cell pools in the testis. This work also demonstrates that production of a secreted Insulin binding protein ImpL2 by CySCs reduces available Dilps in the stem cell niche, and ImpL2 in the niche milieu should reduce insulin signaling in GSCs and CySCs. While these data seemingly contradict the results that insulin is required for GSC maintenance, a model is suggested in which low constitutive levels of insulin signaling are required for stem cell proliferation and that higher levels are required to induce stem cell differentiation. (Amoyel, 2016b).

Prior reports have found that both male and female flies with reduced Insulin or Tor activity are sterile, and the results presented in this study suggest that this is due at least in part to a lack of somatic cell differentiation. The results indicate that Dilp6, the IGF homolog, plays a local role in CySC differentiation, but acts redundantly with other presumably systemic factors, suggesting that both constitutive and nutrient-responsive inputs control CySC differentiation. Indeed, this study shows that in addition to controlling the proliferation of stem cells, systemic insulin is required for their differentiation, as the poorly proliferative Akt1, Dp110 or Tor mutant CySC clones do not differentiate and eventually die by apoptosis. This combination of reduced proliferation and increased apoptosis may explain why other studies suggest that Tor is required for self- renewal in GSCs; indeed prior reports indicate that while Tor mutant GSCs are lost, hyper-activation of Tor leads to faster loss of GSCs through differentiation and recent work indicates that lineage-wide Tor loss blocks the differentiation of GSCs. The use of hypomorphic alleles enabled a genetic separation of the proliferative effects and differentiation requirements of PI3K and Tor in CySCs. Finally, there is evidence that PI3K/Tor activity promotes differentiation of stem cells in gonads in mammals, suggesting that these findings may reflect a conserved role of Tor activity in promoting germ cell differentiation, both through autonomous and non- autonomous mechanisms involving somatic support cells. Moreover, it seems likely that Tor activity may be a more general requirement for the differentiation of many stem cell types, as increased PI3K or Tor has been shown to induce differentiation in many instances. In particular, mouse long term hematopoietic stem cells are lost to differentiation when the PI3K inhibitor Pten is mutated, while Drosophila intestinal stem cells differentiate when Tor is hyperactive due to Tsc1/2 complex inactivation. Moreover, inhibition of Tor activity by Rapamycin promotes cellular reprogramming to pluripotency, while cells with increased Tor activity cannot be reprogrammed, suggesting a conserved role for Tor signaling in promoting differentiated states (Amoyel, 2016b).

The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence has recently been completed, the fertility factors still are not identified, in part because of low cloning efficiency of heterochromatic Y sequences. A method for iterative BLAST searching has been used to assemble heterochromatic genes from shotgun assemblies, and kl-2 and kl-3 have been identified as 1-beta and gamma-dynein heavy chains, respectively. These conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription-PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo. A previously undescribed Y gene, polycystine-related (PRY), was also found. The closest paralogs of kl-2, kl-3, and PRY (and also of kl-5) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes (Carvalho, 2000).

The discovery that the Y chromosome of Drosophila melanogaster contains genes essential only for male fertility dates back to the birth of Drosophila genetics and the theory of chromosomal inheritance. In 1929, Stern showed that these genes are localized in both the short (YS) and long (YL) arms of the Y chromosome, and in 1960, Brosseau used x-ray-induced mutations to identify seven complementation groups, two in YS (ks-1 and ks-2) and five in YL (kl-1 to kl-5). In 1981, Kennison obtained fertile X-Y translocation lines and used them to construct males with deletions in each of the fertility factors. With these lines, Kennison confirmed six of the seven fertility factors previously identified by Brosseau (kl-4 was not confirmed). The same lines allowed a more precise identification of the defects associated with the lack of each of the fertility factors. In particular, the lack of kl-3 or kl-5 causes the loss of the outer arm of the sperm tail axoneme, a structure known to contain the molecular motor protein dynein in other organisms. Indeed, in 1982, Goldstein showed that sperm from kl-3^- and kl-5^- (and also kl-2^-) males lack three discrete high molecular weight proteins with mobility similar to dynein heavy chains of Chlamydomonas reinhardtii and proposed that these fertility factors are the structural genes of three different dynein heavy chain proteins. In 1993, Gepner and Hays sequenced part of kl-5 and showed that it encodes an axonemal beta-dynein heavy chain that is expressed in the testis (Carvalho, 2000 and references therein).

Axonemal dynein heavy chains are known to be responsible for the beating of flagella and cilia, which explains why kl mutants produce immotile sperm. There are several isoforms of axonemal dynein heavy chains (alpha, beta, gamma, 1beta, 1alpha, etc.) that associate to form the inner and outer arms of the axonemes. D. melanogaster has at least seven other dynein heavy chain genes, scattered in chromosomes X, 2, and 3 (Carvalho, 2000 and references therein).

Another important experimental breakthrough was the development of a method to discern banding patterns in Drosophila heterochromatin, which allowed the first detailed cytogenetic investigation of the Y chromosome. Gatti and Pimpinelli (1983) identified 25 heterochromatic bands on the Y and mapped the fertility factors to these bands. It became clear that some of the fertility factors, including kl-5, are unusually large [~3 megabase (Mb)]. The paradox of a conventional coding gene (e.g., kl-5), spread over a huge amount of DNA was solved by Bünemann and coworkers: in the kl-5 homolog of Drosophila hydei, some of the introns are gigantic (>1 Mb) and most likely account for the unusual size of the gene. These introns are composed of short repetitive sequences and satellite DNA. These key discoveries trace back to the extensive work on lampbrush Y chromosomes initiated by Meyer and coworkers in 1961 (Carvalho, 2000 and references therein).

As can be seen from the above summary, the progress on the identification of Y-linked genes has been very slow. This slow progress is mainly a consequence of the technical difficulties caused by the heterochromatic state of the Y chromosome, and most of the experimental breakthroughs mentioned above actually are ingenious ways to implement standard tools used for euchromatic genes in heterochromatin. The Y chromosome does not recombine during meiosis, preventing classical genetic mapping; this problem was solved by Kennison's lines. It does not undergo polytenization, making cytogenetic studies more difficult [solved by Gatti and Pimpinelli]. P element mutagenesis was also more difficult, because the common markers are often silenced when inserted in the Y, but now there are special P constructs that make it possible to overcome this limitation (Carvalho, 2000 and references therein).

The recent sequencing of the Drosophila genome might have yielded the final solution, but again the heterochromatic nature of the Y chromosome posed special difficulties. Most heterochromatin is composed of short repetitive sequences that are not stable in the vectors used in sequencing projects. Thus, despite comprising nearly 30% of the genome, heterochromatic sequences account for only 2% of the sequence reads. Furthermore, its repetitive nature does not allow the assembly of the individual sequence reads (~500 bp) into larger scaffolds, and these into complete chromosome arms. As a result, only 15 kb (a small portion of the kl-5 gene) have been assigned to the Y chromosome, whereas essentially all of the 120 Mb of the euchromatin have been assembled into chromosomes X, 2, 3, and 4. Besides these mapped sequences, 631 scaffolds (ranging from 1 kb to 64 kb, and totaling ~4 Mb of sequence) remain unmapped. These unmapped scaffolds most likely contain pieces of heterochromatic genes, including Y-linked ones (Carvalho, 2000 and references therein).

Of special interest in the study has been the inventive use of iterative BLAST searching for assembly of heterochromatic sequences. The unmapped Drosophila scaffolds (called "armU" in Celera's CD-ROM release of the Drosophila genome) were downloaded from ftp://ncbi.nlm.nih.gov/genbank/genomes/D_melanogaster/, and then an armU database was built by using the FORMATDB program of the STANDALONE BLAST. In this way BLAST searches could be restricted to the set of unmapped scaffolds. In addition to STANDALONE BLAST, extensive use was made of the programs WWWSTANDALONE BLAST (Linux version), NETBLAST, REPEATMASKER (available at http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker), and NAP and GAP2 (available at http://genome.cs.mtu.edu/sas.html). BLAST programs were downloaded from the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (Carvalho, 2000 and references therein).

To see how a Y-linked gene appears in the armU sequences, the complete cDNA of kl-5 (Genbank nucleotide record: AF210453) was used as a query sequence in a BLASTN search against the armU database. In addition to the fragment already identified (CG17616 gene in the Genbank AE002688 scaffold), most of the kl-5 gene was retrieved, scattered across five scaffolds. The majority of these scaffolds contain complete exons (the exception is AE003233, which begins in the middle of an exon). Their 3' ends contain the 5' splice junctions and a variable portion of downstream intronic sequences, often ending with simple repetitive sequences. The 5' ends of scaffolds in armU have analogous structures. Some exons are missing altogether in armU. These observations fit well with the expected behavior of a gene like kl-5 in whole genome shotgun (WGS) projects: exons define unique, nonrepetitive sequences that will be cloned regularly and will be assembled into at least a small scaffold in the end of WGS. Normally sized introns will be readily cloned and assembled along with exons. Indeed, most of the kl-5 scaffolds contain several exons and the intervening short introns. However, some introns of kl-5 probably contain Mega base-sized blocks of repetitive DNA that cannot be assembled by WGS or any other available method. These fragments will rarely be cloned and sequenced and, even if sequenced, would not be assembled into a scaffold. In short, during WGS, a gene like kl-5 will be chopped into several pieces, delimited by the unclonable intronic satellite DNA. Most of the time, a gene immersed in heterochromatin will go undetected by the normal 'first pass' annotation procedures (which rely on gene prediction tools and BLASTX with high stringency), because these methods will work poorly with individual exon sequences. However, the whole gene may be retrieved if a suitable query sequence is available (the kl-5 cDNA in this case) to identify and align its pieces. Very small exons embedded in large introns will most likely be lost during the WGS, and this probably explains the lack of some kl-5 exons (Carvalho, 2000).

To identify other fertility factors in armU, protein sequences were used as query sequences. Suitable proteins were chosen as follows. First the 631 scaffolds of armU were filtered with REPEATMASKER and a BLASTX search of each of them was performed against the nr database (all known proteins, including putative ones) with a rather high stringency (e = 10^-4). There were proteins that gave hits in hundreds of scaffolds; most of them are reverse transcriptases, copia polyprotein, etc., and most likely are matching transposable elements of armU that 'escaped' REPEATMASKER. Some other proteins have hits in a few scaffolds; these are homologs of prospective Y-linked genes, chopped in pieces as kl-5. Each of these prospective proteins was used as a query sequence (as was done with the cDNA of kl-5), running TBLASTN with a lower stringency (e = 10) against the armU database. A staggered pattern results from the very large introns of Y-linked genes. Two such cases are myosin VII (AAF06035, from Dictyostelium discoideum) and gamma-dynein heavy chain, from C. reinhardtii], and were investigated further. All tested scaffolds relating to myosin VII proved to be not Y-linked (i.e., PCR produces bands when either male or female DNA is used as the template), whereas most of the dynein-related were Y-linked. Several of the Y scaffolds were identified by using gamma-dynein heavy chain (Genbank record: Q39575) as a TBLASTN query sequence with a low stringency (sometimes e = 1,000), in an attempt to retrieve missing exons. Because there is a big overlap among several of the dynein-related scaffolds, most likely two different Y-linked dynein heavy chain genes were found (Carvalho, 2000).

Genomic DNA from Y deficient males (kl-1^-, kl-2^-, etc.) was used in PCR to map each of the Y-linked scaffolds identified in the previous step. It should be noted that this procedure assigns a given scaffold to a region of the Y chromosome (e.g., the kl-2 region), but it does not necessarily imply that this scaffold belongs to the actual fertility gene. This distinction is important, because a given region may contain more than one gene. For the sake of simplicity the regions of the Y are referred to by the name of the respective fertility factor they carry (Carvalho, 2000).

Several exons of kl-2 and kl-3 genes are missing from the BLAST results. These missing exons may be absent in armU sequences (as happened with kl-5) or may have diverged enough to be no longer identified by these methods. RT-PCR was used to obtain the sequence of these missing exons and to check whether the Y sequences that were detected are expressed. RT-PCR sequences were obtained from all splice junctions between adjacent scaffolds so that they could be precisely identified. The sequencing of the gaps revealed several previously missed armU scaffolds. In kl-2, some 330 codons of the N terminus are still missing. AE003086 filled the gap between AE003157 and AE002962. There is no sequence gap between AE002962 and AE003049. AE002706 filled a small portion of the gap between AE003049 and AE003219; the remaining 2 kb were sequenced and it was found that it is entirely missing in armU. The AE003219 scaffold contains five internal, short introns and extends through the stop codon. Regarding kl-3, AE002577 and AE002776 appear to be spurious matches caused by running TBLASTN with low stringency, because no RT-PCR product could be recovered that includes these sequences. The gap between AE002917 and AE002920 was sequenced; the 948-bp sequence is missing in armU. Finally, some 230 codons in the C terminus seem to be missing. Each of the three big kl-3 scaffolds contains one internal intron (Carvalho, 2000).

The internal introns were identified and localized with the NAP program, which aligns genomic DNA with proteins allowing for GT/AG bounded gaps (in the case presented here, armU scaffolds and the gamma-dynein Genbank sequence Q39575 were aligned). RT-PCR sequences surrounding each putative intron were obtained and aligned with the corresponding armU scaffold with the GAP2 program, which aligns genomic DNA with cDNA, again allowing for GT/AG bounded gaps. Almost all putative introns suggested by NAP were confirmed, although the inferred splice junctions frequently were not precise. Seven frame-shift sequence errors in armU sequences were pinpointed by NAP and BLASTX and were corrected by sequencing (Carvalho, 2000).

The assembled cDNA of kl-2 and kl-3 were deposited in GenBank under the accession numbers AF313479 and AF313480. Polycystine-related (PRY) is a putative, previously unidentified Y-linked gene. During attempts to retrieve missing exons two armU scaffolds were found that map to the kl-5 region but have no similarity with the kl-5 cDNA. AE002774 seems to contain only two short pieces of transposable elements and was not further investigated. AE003011 showed a strong similarity with the product of a putative gene localized in chromosome 2 (AAF44887) and also a weaker similarity with the human polycystine protein (AAD18021). Interestingly, polycystine is similar to the sea urchin sperm receptor for egg jelly (AAB08448). Using the Drosophila hypothetical protein AAF44887 as a query sequence in TBLASTN (against armU sequences) another closely related scaffold, AE003212 was recovered. RT-PCR closed the gap between it and AE003011; thus, they most likely are part of a previously unidentified expressed Y-linked gene, which is currently being sequenced. Surprisingly, AE003212 maps to the kl-3 region. These findings imply that the breakpoint of the V24 translocation (the h4 band) cuts the PRY gene in the middle. Thus, V24 is defective for PRY -- a close examination of this line may give some clue about the function of this gene. Because the kl-3 and kl-5 regions are known to contain factors (other than the dyneins) that cause sterility when present in three copies, it is possible that PRY is responsible for this phenotype (Carvalho, 2000).

A simple method has been described for tailoring BLAST searches in such a way that poorly assembled fragments from WGS projects may reveal genes embedded in heterochromatin. The method relies on TBLASTN searches (instead of the more usual BLASTX) to identify putative heterochromatic genes by the distinct staggered pattern they produce. It was applied and the kl-2 and kl-3 genes on the Y chromosome of D. melanogaster were successfully identified. The sequences that were found are long, transcribed open reading frames that encode dynein heavy chain polypeptides. Thus the quest for the Y dyneins initiated by Hardy is now finished, and Drosophila Y gene hunting may now move to the even more exciting task of identifying the mysterious kl-1, ks-1, and ks-2 (Carvalho, 2000).

Heterochromatic genes are islands of unique sequence and appear in the end of WGS as isolated scaffolds that could not be assembled into chromosomes. If introns are large enough and contain heterochromatic repeat sequences, they will be sufficiently underrepresented in the WGS sequences to disrupt the assembly of flanking scaffolds. As a result, exons of the same gene are scattered in several unmapped scaffolds ('armU'), generating a staggered pattern in TBLASTN and BLASTN searches. This pattern will occur not only in the Y-linked genes but also in autosomal heterochromatin, as in the case of the Drosophila myosin VII homolog. Although heterochromatic genes pose special problems for genome sequencing, it is possible that they have an advantage over euchromatic genes: once the euchromatic sequence of a whole chromosome arm (a Mb-sized, unique sequence scaffold) is obtained, it may be very difficult to detect all of the genes it contains, whereas it is possible that a significant proportion of the unique heterochromatic sequences represents functional genes. It seems that only functional sequences resist the forces that fill heterochromatic regions with short repeats and thus remain clonable, unique sequences. During this project, 38 armU scaffolds were examined, and at least 12 of them (~1/3) seem to be part of genes. Of course, this sample is not random, but it strongly suggests that the small isolated scaffolds remaining at the end of WGS projects may be a good source of interesting genes. Indeed, it is an advantage of the WGS approach over clone-based strategies that, besides the euchromatin, it also retrieves unique sequence heterochromatin, no matter how deeply the sequence is located within the heterochromatin (Carvalho, 2000).

A striking pattern emerges from the phylogeny of the Y dyneins: they all are closely related to other Drosophila genes, but none of these paralogous genes is X-linked. The same pattern occurs with PRY. Furthermore, the Drosophila X chromosome contains only one dynein heavy chain (Dhc 16F), in contrast with the three Y-linked ones. Thus, it seems likely that these genes were acquired from autosomes, rather than being present in the hypothetical chromosome pair that gave rise to the X and Y. This mechanism has been demonstrated for the mammalian Y, but in that case the Y chromosome also exhibits a number of X-derived genes. Another Drosophila Y-linked gene, Su(Ste), has been shown to be recently originated from an autosomal gene. Repetitive sequences also do not show any sign of X-Y homology; it has been proposed that much of the Drosophila Y is virtually a new construct, rather than a degenerated X. The data presented in this study clearly support this hypothesis. It remains to be seen whether any part of the ancestral Y was homologous to the X (as may be the case for rDNA genes, which are present in both X and Y chromosomes) or whether it is a totally new construct. This picture of the Drosophila Y may change if other, yet unidentified Y genes (kl-1, ks-1, ks-2, etc.) turn out to have X homologs. Whatever its origin, the present configuration of the Drosophila Y chromosome seems to be quite old, for at least kl-5 is present also in D. hydei and Drosophila mediopunctata, which diverged from D. melanogaster ~39 million years ago. A few Drosophila species have fertile X0 males; it will be most interesting to study the location of their axonemal dynein heavy chain genes (Carvalho, 2000).

The absence of X homologs and the close similarity between Y and autosomal genes suggest that the former is an agglomeration of autosomal genes. This hypothesis is the most parsimonious and explains well the kl-5, kl-3, and PRY cases. However, it is also possible that Y chromosomal genes have transposed to the autosomes, and this possibility might explain the kl-2 case. The closest paralogs of kl-3 and kl-2 are the CG9492 and CG9068 genes, respectively. Dynein heavy chains have ~4,500 amino acids, whereas CG9492 and CG9068 are shorter (3,508 and 1,227, respectively) and seem to lack the C terminus. The former case results from a misannotation: BLASTX and NAP identified all of the missing ~1,000 amino acids of CG9492 (including the stop codon at position 188,944 in the AE003683 scaffold). However, CG9068 seems to be truncated, for no sign of the 'missing' C terminus could be found. Therefore, the relationship between CG9068 and kl-2 is unclear; it is possible that kl-2 originated from CG9068 and that after this the latter suffered a deletion. However, it is also possible that CG9068 results from a partial transposition (perhaps being a pseudogene) of kl-2 (Carvalho, 2000).

The phylogeny of the dyneins strongly suggests that kl-2 encodes a 1beta-dynein, whereas kl-3 encodes a gamma-dynein. This phylogeny fits well with the known mutant phenotypes of kl genes and with the function of dynein heavy chains; kl-3^- mutations (but not kl-2^-) disrupt the outer arms of axonemal microtubules, and gamma-dyneins are part of these structures. 1beta-dyneins are part of the inner arms, and it remains to be explained why kl-2^- mutants do not show cytological defects (Carvalho, 2000).

It has been noted that the human Y chromosome exhibits a 'functional coherence'; besides housekeeping genes, many Y genes have male-related functions, which contrasts with the random content of the other chromosomes. It is striking that the Drosophila Y has an even stronger coherence, approaching obsession; all known fertility factors (kl-2, kl-3, and kl-5) encode proteins belonging to the same gene family (axonemal dynein heavy chain). This extreme functional coherence, coupled with the lack of X homologs (which might provide an 'historical' cause), begs for an explanation (Carvalho, 2000).

Theoretically, the Y chromosome is expected to accumulate male-related genes; male-female antagonistic effect of genes may hamper the evolution of male-related traits, unless they are located in a male-specific region of the genome. This prediction has been demonstrated experimentally, and the findings presented here support it. Regarding the particular male fitness trait involved, the most likely advantage conferred by sperm axonemal motor proteins is sperm competitive ability. The PRY gene may also be involved in sperm competition if it has a function similar to its homolog in sea urchin. Drosophila females mate several times; thus, there is ample room for sperm competition, and clearly there is genetic variation for this trait. It is proposed that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes, most likely caused by sperm competition. This hypothesis explains the puzzling finding of a Y chromosome packed with motor proteins which are absent in the X chromosome. The large element of chance involved in the occurrence of the appropriate translocations probably explains the apparent incompleteness of the process, that is, outer arms are composed of alpha-, beta-, and gamma-dyneins, but only beta and gamma got Y counterparts (Carvalho, 2000).

The hypothesis that natural selection has driven an accumulation on the Drosophila Y of genes related to sperm function may be tested in several ways. (1) Studies designed to quantify Y-linked variation in sperm competition are clearly needed. (2) The comparative method of looking for dynein heavy chain genes in other Diptera (including species with fertile X0 males) may reveal the intermediate steps of the birth of dynein-packed Y chromosomes. (3) the identification of the other fertility factors may yield more clues about the forces shaping Y chromosome evolution in Drosophila (Carvalho, 2000).

In conclusion, there is significant transcription from several genomic loci in Drosophila spermatids, and the post-meiotically expressed transcripts localise to the growing ends of spermatids. This transcription and RNA localisation occurs before spermatid chromatin remodelling, and at least one post-meiotically-expressed gene is required for spermiogenesis (Barreau, 2008).

Mutation in S-phase cyclin A-associated protein in the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. This study generated and analysed short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding was assayed by total internal reflection fluorescence microscopy. Patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. It was further shown that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and ssp3 directly bind MTs. These results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis (Wormser, 2020).

CLP1, TSEN complex, and VCP are evolutionarily conserved proteins whose mutations are associated with neurodegenerative diseases. This study found that they are also involved in germline differentiation. To optimize both quantity and quality in gametes production, germ cells expand themselves through limited mitotic cycles prior to meiosis. Stemming from previous findings on the correlation between mRNA 3'-processing and meiosis entry, this study identified that the RNA kinase Cbc, the Drosophila member of the highly conserved CLP1 family, is a component of the program regulating the transition from mitosis to meiosis. Using genetic manipulations in Drosophila testis, this study demonstrates that nuclear Cbc is required to promote meiosis entry. Combining biochemical and genetic methods, it is revealed that Cbc physically and/or genetically intersects with Tsen54 and TER94 (VCP ortholog) in this process. The C-terminal half of Tsen54 is both necessary and sufficient for its binding with Cbc. Further, this study illustrates the functional conservation between Cbc and mammalian CLP1 in the assays of subcellular localization and Drosophila fertility. As CLP1, TSEN complex, and VCP have also been identified in neurodegenerations of animal models, a mechanism involving these factors seems to be shared in gametogenesis and neurogenesis (Jianbo, 2021).

Germ cells undergo distinct nuclear processes as they differentiate into gametes. While these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly-conserved Importin β/karyopherin family member Importin-9 (Ipo9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization (FISH) revealed that Ipo9 (KO) mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9 (KO) mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis (Palacios, 2021).

Subcellular compartmentalization allows for complex modes of gene regulation in eukaryotic cells. The regulated and active transport of macromolecules between different compartments promotes cellular homeostasis and often drives differentiation. Transport of molecules from the cytoplasm to the nucleus depends on a family of proteins called karyopherins, also known as importins. The karyopherin superfamily of transporters consists of importin α and importin β subgroups. All the proteins within this karyopherin superfamily share tandem huntingtin, elongation factor 3, protein phosphatase 2A and mechanistic target of rapamycin (HEAT) repeats. These repeats allow these proteins to bind to various cargo proteins, which often, but not always, contain a nuclear localization signal within their peptide sequence. Karyopherins then transport these cargoes into the nucleus through nuclear pores (Palacios, 2021).

Another key component of the transport machinery is the small GTPase Ran (Cautain, 2015). Cytoplasmic Ran is typically maintained in a GDP-bound state, whereas nuclear Ran binds GTP. This concentration gradient of GDP and GTP bound Ran provides a directional cue for the transport of proteins between the cytoplasm and nucleus. Once importins enter the nucleus, high affinity interactions with RanGTP cause karyopherins to release their cargoes and recycle back to the cytoplasm (Palacios, 2021).

Accumulating evidence suggests that β-karyopherins do not simply function as constitutive and redundant housekeeping proteins. Interactions between different β-karyopherins with specific cargoes depend not only on their overlapping expression patterns in time and space, but also on clear differences in the affinities of the physical interactions. For example, histones can bind to multiple β-karyopherins, but their affinities vary. For example, Kapβ2 and Imp5 exhibit very strong affinity for Histone H3, whereas Impβ, Imp4, Imp7, Imp9 and Impα display weaker interactions. Additionally, a previous study identified a group of 468 cargoes for 12 β-karyopherins. Three hundred and thirty two of these cargoes were unique to one β-karyopherin family member, suggesting a division of function among these transporters. Several β-karyopherin family members have been associated with specific diseases. Accumulating evidence shows that β-karyopherins are overexpressed in multiple tumors including melanoma, pancreatic, breast, colon, gastric, prostate, esophageal, lung cancer and lymphomas. Additionally, specific karyopherin-β proteins, such as exportin-1, have been implicated in drug resistance in cancer (Palacios, 2021).

Many importins exhibit enriched expression in gonads and are functionally required during different stages of spermatogenesis and oogenesis across many species, including Drosophila. Drosophila ovaries are organized into discrete units called ovarioles, which contain a series of sequentially developing egg chambers. Each egg chamber is comprised of 16 germ cells, 15 nurse cells and one oocyte, surrounded by a layer of somatic follicle cells. The initiation of meiosis occurs early in oogenesis, marked by the formation of the synaptonemal complex (SC) and the generation of the programmed double strand breaks. After these first events, oocytes remain arrested in prophase 1 of meiosis until stage 12, followed by prometaphase 1 at stage 13 and metaphase 1 at stage 14 (Palacios, 2021).

The Drosophila testis is structured as a closed-end coiled tube. At the tip of the testis, 10-14 germline stem cells (GSCs) surround a small cluster of somatic cells called the hub. GSCs typically divide asymmetrically to produce another GSC and a gonialblast. Gonialblasts become enveloped by two somatic cyst cells, that function in an analogous manner to the Sertoli cells of the mammalian testis. The Drosophila gonialblast goes through four incomplete mitotic divisions to form an interconnected 16-cell spermatogonial cell cyst. Each spermatocyte within the cyst undergoes meiosis, resulting in the formation of cysts that contain 64 interconnected haploid cells. Immediately after the completion of meiosis, these cells enter the 'onion stage', which is marked by the appearance of the nebenkern, a two-stranded helical structure derived from mitochondria. Defects in meiosis can result in the appearance of fragmented nebenkern and alterations in the normal 1:1 ratio of nuclei and nebenkern (Palacios, 2021).

Spermiogenesis is marked by nuclear elongation and chromatin reorganization. Nuclear elongation is dependent on microtubules from the basal body that associate with the nucleus. Chromatin organization switches from a histone-based to protamine-based packaging in the late elongation stage. During elongation, the nuclear envelope that is in contact with the basal body forms a cavity that fills with microtubules while the nucleus takes on a 'canoe' shape. During chromatin reorganization, histones are ubiquitylated by an unknown ubiquitin ligase and subsequently degraded by the proteasome at the late canoe stage, immediately before protamines are incorporated into the chromatin. After histone removal, the transition like-protein (Tpl) is incorporated. This facilitates protamine incorporation. In Drosophila, mature sperm contain Mst35Ba (protamine A), Mst35Bb (protamine B) and Mst77F. Towards the end of spermiogenesis, sperm form their own membranes in a process called individualization (Fabian and Brill, 2012) (Palacios, 2021).

In Drosophila, mutants in several importins develop normally into adults, but exhibit various defects in fertility. Importin α2 mutant males exhibit a dramatic decrease in the formation of individualized and motile sperm, whereas mutant females produce small and deflated eggs with missing or fused dorsal appendages. Similarly, mutations in Importin α1 also cause male and female sterility, marked by egg-laying defects in females and the formation of spermatocytes with abnormally large round nuclei in males, and loss of Importin α3 leads to the arrest of oogenesis. The specific cargoes responsible for these phenotypes remain unknown (Palacios, 2021).

This study reports that null mutations in Ipo9 (also known as Ranbp9) cause disruption of chromosome segregation and condensation during meiosis in both female and male Drosophila. Previous results have shown that Ipo9 helps to traffic Actin, Histone H2A-H2B dimers and a variety of other factors into nuclei. This study confirmed that loss of Drosophila Ipo9 disrupts the accumulation of nuclear actin during oogenesis. In addition, it was found that Ipo9 promotes chromosome segregation during meiosis, and the exchange of histones for protamines during spermiogenesis. Biochemical experiments suggest that Ipo9 physically associates with proteasome components, and immunofluorescence studies show that loss of Ipo9 disrupts the normal trafficking of the proteasome into germ cell nuclei during spermiogenesis. Together, these data reveal new processes directly regulated by a specific nuclear transport factor during gametogenesis (Palacios, 2021).

This study provides evidence that Ipo9 specifically regulates a number of critical processes during Drosophila gametogenesis. Ipo9 null mutants survive to adulthood but exhibit female and male sterility. In the ovary, loss of Ipo9 results in defects in chromosome orientation and segregation during meiosis, resulting in mitotic catastrophes during early embryogenesis in progeny derived from Ipo9 homozygous mutant females. Ipo9 mutant males also exhibit numerous phenotypes during germ cell development, including defects in meiosis, and disruption of the nuclear shape changes and failure to fully exchange histones for protamines during spermiogenesis. Together, these represent a unique spectrum of phenotypes compared to other Drosophila β-karyopherin family members. Of the 12 Drosophila β-karyopherin genes that have been genetically characterized, loss-of-function alleles in seven result in lethality. Several other importin mutants do survive until adulthood, including ebomut, aplnull and artsnull. ebomut homozygotes display neuronal defects, aplnull mutants are male sterile, whereas artsnull mutant females produce smaller eggs that cannot be fertilized. In addition, a ketel dominant negative mutant (ketel^D) shows a female sterile phenotype and embryos derived from these flies exhibit chromosome segregation defects somewhat similar to those displayed by Ipo9KO mutants. Thus, amongst Drosophila karyopherin family members studied to date, Ipo9 is the only gene that displays specific defects during meiosis in both females and males, and in late sperm development, when mutated (Palacios, 2021).

Transgenic rescue experiments confirm that Ipo9 functions to promote the transport of proteins from the cytoplasm to the nucleus during oogenesis and spermatogenesis. A full-length Ipo9 transgene rescues most of the sterile phenotypes exhibited by Ipo9 mutants when driven in the germline. It is suspected that the failure of the Ipo9 wild-type transgene to fully rescue the male sterility of the mutant is likely due to the failure of the vasa-gal4 driver to fully recapitulate the endogenous expression pattern of Ipo9. The N-terminal domains of β-karyopherin proteins normally promote cytoplasmic-to-nuclear trafficking by contacting the nuclear pore and helping cargoes move through the nuclear pore complex. This domain also binds to RanGTP, and thus participates in the cycling of importins back-and-forth between the cytoplasm and nucleus. Strikingly, deletion of the N-terminal karyopherin domain renders the transgene non-functional, confirming that Ipo9 acts as an essential transport factor during gametogenesis in both males and females (Palacios, 2021).

The transition from histone-based to protamine-based chromatin organization is essential for the nuclear shaping that leads to a highly compact sperm nucleus. Ipo9KO nuclei are able to incorporate protamine-B, however histone H2A and H2Av are not completely removed. These results may partially explain why Ipo9KO nuclei do not elongate properly. Evidence that the ubiquitin proteasome pathway is involved in histone removal during spermiogenesis includes the histone ubiquitylation that occurs before protamine deposition and the delay in histone removal in proteasome mutants. Interestingly, Ipo9KO nuclei do not exhibit strong nuclear ubiquitylation after protamine incorporation, even though they still retain nuclear histones. Additionally, it was observed that Ipo9KO spermatids showed a significant reduction in the nuclear localization of several proteasome proteins, including Prosα6T, Prosα3T and Prosα2, compared to the control spermatids. These results suggest that the ligase(s) responsible for histone ubiquitylation and components of proteasome that ultimately degrade ubiquitylated histones are potential cargoes of Ipo9. Interestingly, Ipo9 appears to physically associate with a number of specific ubiquitin ligases, including Hyperplastic discs and KLHL10, which have been implicated in the regulation of male germ cell development, and with several components of the proteasome. Perhaps Ipo9 has evolved to temporally coordinate the import of these functionally related proteins during late sperm development. Such specialization in nuclear import may offer an economy of scale that would not exist if the responsibility of nuclear import during this critical phase of sperm development, when the cytoplasm and nuclei of sperm are becoming highly compacted, was spread across a number of potentially redundant β-karyopherins. This type of coordination in trafficking has been proposed previously in different contexts. Thus, further study of Ipo9 cargoes during sperm development may reveal critical unknown factors that play roles in meiosis, chromosome compaction and segregation, and nuclear shape changes (Palacios, 2021).

Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recent work has shown that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, this study reports that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging (Butsch, 2022).

Gene expression evolution is typically modeled with the stochastic Ornstein-Uhlenbeck (OU) process. It has been suggested that the estimation of within-species variations using replicated data can increase the predictive power of such models, but this hypothesis has not been fully tested. This study developed EvoGeneX, a computationally efficient implementation of the OU-based method that models within-species variation. Using extensive simulations, this study showed that modeling within-species variations and appropriate selection of species improve the performance of the model. Further, to facilitate a comparative analysis of expression evolution, a formal measure of evolutionary expression divergence was introduced for a group of genes using the rate and the asymptotic level of divergence. With these tools in hand, this study performed the first-ever analysis of the evolution of gene expression across different body-parts, species, and sexes of the Drosophila genus. It was observed that genes with adaptive expression evolution tend to be body-part specific, whereas the genes with constrained evolution tend to be shared across body-parts. Among the neutrally evolving gene expression patterns, gonads in both sexes have higher expression divergence relative to other tissues and the male gonads have even higher divergence than the female gonads. Among the evolutionarily constrained genes, the gonads show different divergence patterns, where the male gonads, and not the female gonads, show less constrained divergence than other body-parts. Finally, interesting examples were shown of adaptive expression evolution, including adaptation of odor binding proteins (Pal, 2023).

It was hypothesized that, in addition to the somatic silencing of testis-specific gene clusters, LamDm₀ is also required for attachment of these clusters to the nuclear envelope. To test this hypothesis, intranuclear positions of the 60D1 and 22A1 regions was determined in the interphase nuclei of cultured S2 cells in which LamDm₀ was ablated by RNAi. Fluorescence in situ hybridization (FISH) combined with immunostaining for LamDm₀ confirmed RNAi-induced ablation of LamDm₀, and showed approximately 2-fold decrease in frequency of the 60D1 and 22A1 loci bound to lamina. Next, association of the 60D1 gene cluster with nuclear envelope wa analyzed during male germ-line differentiation. The whole-mount testes dissected from the third instar larvae were analyzed for intranuclear localization of the 60D1 region by FISH combined with immunostaining for LamDm₀. Morphologically, a group of small cells is located at the end of testis and includes spermatogonia, cyst cells and stem cells, in which the 60D1 locus is silent. In these cells, the 60D1 region is associated with nuclear lamina in 76% of nuclei, similarly to the cultured somatic S2 cells. On the contrary, in spermatocytes (identified by characteristic large nuclei), in which testis-specific genes in the 60D1 region are expressed, this region is found at the nuclear envelope in only 6% of the nuclei. Thus, transcriptional activation of the testis-specific gene cluster in male germ line is coupled to its dissociation from the nuclear envelope. Similarly, detachment from the nuclear lamina has been associated with transcriptional activation of other lamina-bound loci both in Drosophila and in mammals. These observations strongly suggest a model in which gene repression is controlled in a cell type-specific manner through regulated tethering of chromatin to the nuclear lamina. Further dissection of the mechanisms that mediate repression of lamina-bound multigenic regions and control localization of these regions at nuclear envelope will provide new insights into coordinated regulation of tissue-specific genes, thus advancing understanding of cell differentiation both in normal development and in disease (Shevelyov, 2009).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes (see Model for the developmentally programmed steps that oppose PcG repression and turn on terminal differentiation gene expression). In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells here did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TF_IID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TF_IID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TF_IID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression (Ezhkova, 2009), and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

During the final stages of spermatogenesis in Drosophila, as in many other invertebrate and vertebrate species, chromatin reorganization events cause the spermatid nuclei to become tightly compacted. Histones associated with spermatocyte chromatin are ultimately exchanged for protamines, allowing the nucleus to condense up to 200 fold. Two genes encoding protamines have been identified in Drosophila (Mst35Ba, or Protamine A; and Mst35Bb, or Protamine B). Additionally, the gene Tpl94D demonstrates functional homology to mammalian transition proteins, which bind chromatin as an intermediate step between histone-based and protamine-based chromatin organization. Therefore, nuclear compaction in Drosophila occurs as a two-step process: histones are first displaced by transition proteins, and transition proteins are later exchanged for protamines (Sartain, 2011 and references therein).

Soon after protamine incorporation, the spermatids in a cyst become separated from one another in a process called individualization. During this process, a cone-like structure composed of cross-linked F-actin assembles around each nucleus in the cyst. The 64 cones in the cyst move as a unit down the length of the sperm tails, simultaneously pushing out excess cytoplasm and wrapping each spermatid in an individual membrane. The separated, mature sperm then roll into coils and exit the testis to be stored in the seminal vesicle (Sartain, 2011).

In Drosophila, there are many examples of transcripts that are synthesized in spermatocytes but are not translated until after meiosis, to such an extent that transcriptional activity in the developing Drosophila sperm cell was previously thought to be predominantly limited to early spermatocytes and spermatogonia. However, recent evidence demonstrates that transcriptional activity occurs post-meiotically as well. For those transcripts that remain quiescent until post-meiotic stages, a translational control mechanism must be in place (Sartain, 2011).

Many cell types, including oocytes and neurons, achieve translational regulation through adjusting the length of the poly(A) tail in the cytoplasm. A long poly(A) tail promotes translation of the transcript through recruitment of translation initiation factors, whereas a transcript with a short poly(A) tail remains untranslated or is degraded. Most mRNAs are extensively polyadenylated in the nucleus; however, for some transcripts that will be held in an untranslated state for a period of time, poly(A) tail modifications occur outside the nucleus. In Xenopus, transcripts destined for post-transcriptional poly(A) tail adjustment contain two consensus sequences in their 3'UTR: a cytoplasmic polyadenylation element (CPE) and the hexamer AAUAAA; these recruit a complex of proteins that alter poly(A) tail length. In Xenopus, CPE is bound by CPE-binding protein (CPEB). The cleavage and polyadenylation specificity factor (CPSF) binds to the hexamer. CPEB and CPSF recruit a cytoplasmic poly(A) polymerase (PAP) and a deadenylase, both of which work on the transcript simultaneously. However, the deadenylase is slightly more efficient than the PAP, so the net effect is a poly(A) tail that remains short. Upon a signal to activate translation, CPEB is phosphorylated, causing the deadenylase to dissociate from the complex; the PAP is then free to elongate the poly(A) tail (Sartain, 2011).

PAPs that act in the cytoplasmic complex differ from nuclear PAPs. The Gld-2 (germline development 2) family of cytoplasmic PAPs has been described in C. elegans, Xenopus and Drosophila. Whereas nuclear PAPs contain a catalytic domain and an RNA-binding domain, Gld-2 family members have only a catalytic domain. For RNA specificity, Gld-2 associates with an RNA-binding protein, typically a Gld-3, to form a heterodimer that acts as a cytoplasmic PAP (Sartain, 2011 and references therein).

Gld-2 family members have been shown to play roles in oogenesis in several organisms. In worms, a Gld-2 homolog is involved in the mitosis/meiosis decision to make both male and female germ cells (Kadyk, 1998). In Drosophila, the X-linked Gld-2 homolog wispy (wisp) is necessary for oogenesis and egg activation. WISP is present in ovaries but not testes and is necessary for the completion of meiosis in oocytes. WISP has been shown to polyadenylate transcripts of cortex, which is required for proper meiotic progression. WISP also polyadenylates several developmental transcripts, the protein products of which are needed for early embryogenesis, including bicoid, Toll and torso (Sartain, 2011 and references therein).

The Drosophila genome contains an autosomal paralog of wisp called Gld2. Previous studies of Gld2 have demonstrated a role in long-term memory and show that GLD2 acts as a PAP in vitro. This study shows that Gld2 is expressed in the male, but not female, germline. It is required for the completion of spermatogenesis, specifically for the elongation and individualization stages. In GLD2 knockdown testes, the first disruption observed is post-meiotic, at the onset of spermatocyst elongation. In these testes, the nuclei in developing cysts scatter and basal bodies are not observed near nuclei. F-actin-containing individualization complexes do not assemble and nuclear compaction does not complete. Additionally, protamines are not incorporated and transcripts for both dynamin (shibire - FlyBase) and the transition protein are undetectable. These findings indicate that Gld2 arose from duplication of the wisp locus, and that this derived paralog was likely maintained in the genome owing to its essential role in spermatogenesis (Sartain, 2011).

The genus Drosophila can be divided into subgenera, and genome sequences are available for two of these: Sophophora (which contains D. melanogaster) and Drosophila (which contains species that diverged from D. melanogaster ~63 million years ago). wisp and Gld2 orthologs have been identified in species from both subgenera. Available gene expression data show that the wisp orthologs in D. melanogaster, D. mojavensis and D. virilis have female-biased expression, whereas the Gld2 orthologs from D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. pseudoobscura and D. mojavensis have male-biased expression). This is consistent with the conservation of the oogenesis and spermatogenesis functions of WISP and GLD2, respectively, across the entire genus. All other insect species with sequenced genomes (including Anopheles gambiae and Aedes aegypti, which are the closest relatives to Drosophila with completely sequenced genomes) possess only a single ortholog of wisp/Gld2, based on best reciprocal BLAST searches. Therefore, a duplication event occurred after the most recent common ancestor (MRCA) of Drosophila and mosquitoes, but prior to the MRCA of the genus Drosophila, to give rise to either wisp or Gld2 (depending on which locus is ancestral and which is derived). A phylogenetic reconstruction of the evolutionary relationships of the insect protein coding sequences supports this hypothesis. Furthermore, the wisp/Gld2 ortholog in A. gambiae has female-biased expression, based on microarray data from whole males and females. This has led to a hypothesis that the ancestral germline function of this gene family is in ovaries, and that the testis function of GLD2 is derived (Sartain, 2011).

The testis-expressed cytoplasmic PAP, GLD2, is required for spermatogenesis in D. melanogaster. Knockdown of GLD2 in the testes causes widespread defects in post-meiotic spermatogenesis events. In a GLD2 knockdown, the earliest defects are seen in early post-meiotic spermatids, when the basal body fails to dock at the nuclear envelope and the nuclei begin to scatter. Many late-stage events of spermatogenesis are also affected, including protamine translation and F-actin cone formation on individualization stage spermatids. Additionally, GLD2 knockdown affects the stability of dynamin transcripts and those of transition protein (Tpl94D) in the testes. Interestingly, GLD2-deficient germ cells appear to undergo normal meiosis, in contrast to mutants of other Gld-2 homologs, including the GLD2 paralog WISP (Sartain, 2011).

There is indirect evidence that GLD2 acts as a cytoplasmic PAP in the Drosophila testes. The GLD2 protein contains a PAP/25A domain, which is shared by all known Gld-2 family proteins. Additionally, GLD2 has the ability to elongate poly(A) tails in vitro. The current study has shown that GLD2 interacts with the Gld-3 homolog BIC-C in a yeast two-hybrid assay. Furthermore, at least two transcripts are absent in GLD2 knockdown testes, which might be the result of destabilization owing to an inability to elongate their poly(A) tails. Taken together, it is concluded that Drosophila GLD2 does act as a PAP during spermatogenesis and that the defects seen in its absence are the result of failure of one or more polyadenylation events (Sartain, 2011).

GLD2 affects many aspects of spermatogenesis in Drosophila. First, nuclear anchoring and basal body docking are defective in the absence of GLD2. Both processes occur in early post-meiotic stages of spermatogenesis in wild-type testes; however, in the absence of GLD2, spermatid nuclei scatter throughout spermatogenic cysts and basal bodies cannot assemble at the nuclear envelope. It is possible that, in the GLD2 knockdown, the failure of these events is related. For example, it might be the case that nuclear anchoring cannot occur until basal bodies have docked properly, or vice versa. Alternatively, loss of GLD2 might affect formation of the post-meiotic nuclear envelope, which might in turn have negative effects on both nuclear anchoring and basal body docking processes. Other studies of mutants that involve basal body defects have documented nuclear localization disruptions, indicating that these two processes may be linked (Sartain, 2011).

Second, GLD2 knockdown testes show abnormalities during nuclear condensation: nuclei clearly begin to condense, but condensation stalls at the canoe stage and does not progress further. It is believed that this phenotype reflects, at least in part, the absence of Tpl94D transition protein transcripts in GLD2 knockdown testes, and thus the inability of nuclei to progress to a condensation state at which protamines would be incorporated. In addition, and perhaps contributing to the phenotype, Protamine B is not translated in the absence of GLD2, even though its RNA is present. Thus, GLD2 acts upstream of Protamine B translation. Given that no effects were detected of GLD2 knockdown on the poly(A) tail length of protamine transcripts, it seems likely that removing GLD2 causes a block in the spermatogenic developmental pathway at a stage before protamine transcripts would normally be translated. Rathke and colleagues showed that protamines are incorporated into the spermatid chromatin after the onset of transition protein incorporation, so it is possible that a lack of transition protein causes a developmental block in GLD2 knockdown testes and that the lack of protamine translation in these testes reflects this block. There is evidence that protamine transcripts are translationally repressed for a few days after their transcription and that this repression is dependent upon elements in their 5'UTR. Thus, GLD2 could be responsible for controlling the translation of a crucial element that causes the relief of repression at the protamine 5?UTR, while not affecting the poly(A) tail status of the protamine transcript itself (Sartain, 2011).

A third defect in GLD2-deficient spermatogenesis occurs at individualization: actin cones are never detected around late-stage nuclei and the spermatids do not separate from one another. dynamin transcripts are missing in GLD2 knockdown testes. The absence of Dynamin could account for the lack of actin cones at individualization stage nuclei: previous studies have demonstrated that Dynamin is present throughout the actin cones and that disruption of Dynamin function in temperature-sensitive mutants contributes to their instability. Lack of dynamin mRNA could indicate that it is a GLD2 target: lack of dynamin polyadenylation by GLD2 could leave the transcript vulnerable to exonucleases in the cytoplasm, resulting in its degradation. Alternatively, it is possible that the absence of Dynamin in the GLD2 knockdown results from a developmental block during late spermatogenesis, at a time before dynamin RNA would be present (Sartain, 2011).

GLD2 localization might help to identify its target transcripts. Immunofluorescence staining experiments showed that in addition to cytoplasmic localization in spermatocytes, GLD2 localizes to the distal ends of elongated spermatogenic cysts. This is where the polarized growth of the cyst occurs in accordance with axoneme extension; additionally, a group of mRNAs that are transcribed post-meiotically have been shown to localize to the distal end of the spermatogenic cyst. Interestingly, one of these late-transcribed genes is orb, which encodes the Drosophila ortholog of CPEB, the protein necessary for cytoplasmic polyadenylation in Xenopus. The presence of both the CPEB ortholog ORB and the cytoplasmic PAP GLD2 at the end of the cyst where growth is occurring might indicate an involvement of GLD2 in late spermatocyst growth. Taken together, these data suggest that the distal end of the cyst might be a major production center for cyst growth, with the necessary mRNAs regulated post-transcriptionally through cytoplasmic polyadenylation (Sartain, 2011).

This study has shown that GLD2 plays an essential for male, but not female, gametogenesis. This is a unique finding among the literature describing other Gld-2 homologs, where Gld-2 proteins are necessary for some aspect of oogenesis and egg maturation in Drosophila, Xenopus and mice and for the proliferative stages of gametogenesis in hermaphrodite worms. Drosophila GLD2 plays a role in the male, but not female, germline, and is required in spermatid morphogenesis rather than in proliferative stages. The evidence that Gld2 was retrotransposed to the third chromosome from a duplication of the wisp locus on the X chromosome might give insight to how this unique role for a Gld-2 homolog came about. The phenomenon of meiotic sex chromosome inactivation (MSCI) might have contributed to duplication of the wisp gene and to subsequent retention of Gld2. During spermatogenesis in Drosophila and other animals, the X chromosome is transcriptionally silenced prior to autosomal silencing. Therefore, genes located on the X chromosome have a limited capacity to encode proteins involved in spermatogenesis. Interestingly, an excess of genes has been retrotransposed from the X to the autosomes, and the autosome-derived copies are hypothesized to allow for the escape from X-inactivation. The testis-biased expression and spermatogenic functions of Gld2 suggest that it was selectively retained because it performs a function unavailable to wisp because of MSCI (Sartain, 2011).

It is interesting that Gld2 is crucial for post-meiotic spermatogenesis in Drosophila, whereas all Gld-2 family members analyzed so far in Drosophila and other species play roles specifically at meiosis. It is hypothesized that the function of GLD2 in the male germline reflects its evolutionary origin: duplication of the X-linked wisp locus allows for an autosomal copy that can be expressed in the testis during MSCI. Although this is the first example of a Gld-2 family member with its gametogenic role solely in spermatogenesis, other species might have developed similar mechanisms of translational control in the testes; for example, spermatogenesis in mice is regulated, in part, by a cytoplasmic PAP outside of the Gld-2 family called TPAP (PAPOLB - Mouse Genome Informatics). Further investigation and identification of GLD2 targets in Drosophila testes will help to elucidate how spermatogenesis can be regulated through cytoplasmic polyadenylation (Sartain, 2011).

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little of this process is known for Drosophila. This study characterizes three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame (Raja, 2005).

For mammals, the somatic set of histones are modified, as these are in part replaced by specific variants during meiotic prophase. After meiosis, histones are replaced by major transition proteins TP1 and TP2 and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements leads to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing protamines as major chromatin condensing proteins and DNA. Some mammals have only one protamine gene, while mice and humans have two genes encoding two different protamines, both of which are essential for fertility and are haploinsufficient. HILS1 (spermatid-specific linker histone H1-like protein) has been proposed to participate in chromatin remodeling in mouse and human spermiogenesis. The transition between histone removal and its replacement by protamines in mice and humans is characterized by small 6- to 10-kDa transition proteins acting as a short-term chromosomal proteins. In mice, the transition proteins TP1 and TP2 are redundant in function. In fishes and birds, transition proteins are missing and protamines directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal configuration is maintained in sperms, while protamine-like proteins have been described for mussels. These protamine-like proteins lack the typical high cysteine content necessary for disulfide bridges. Therefore, a doughnut-type chromatin structure as in mammals is unlikely to occur in mussels. It has been proposed that the protamine-like proteins in mussels belong to the histone H1 family. The sperm chromatin of mussels contain core histones and thus a nucleosomal configuration, but histone H1 is replaced by protamine-like molecules which organize the higher order structure of the chromatin (Raja, 2005).

For Drosophila melanogaster, chromatin reorganization after meiosis has not been studied at the molecular level. At the light microscopic level, the Drosophila spermatid nucleus is initially round after meiosis and then is shaped to a thin needle-like structure with highly condensed chromatin, so that the volume of the nucleus is condensed over 200-fold. In mammals, the volume of the nucleus is reduced over 20-fold. In the mature sperms of Drosophila, core histones are not detectable by immunohistology. There is histochemical evidence for the presence of very basic proteins in sperm, but it still remains an open question whether histones are replaced by protamine-like basic proteins in Drosophila. The analysis of the Drosophila genome sequence revealed that the proteins encoded by two genes show similarity to mammalian protamines for which the male-specific transcripts Mst35Ba and Mst35Bb have been found and have been proposed to encode protamine-like proteins. Another male specifically transcribed gene, Mst77F, is a distant relative of the histone H1/H5 (linker histone) family and has been proposed to play a role either as a transition protein or as a replacement protein for compaction of the Drosophila sperm chromatin. With enhanced green fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and Mst77F encodes a linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male pronucleus requires the function of the maternal component, Sesame, but not for the removal of Mst77F. It has been shown that sesame mutants cause impairment of the entry of histones into the male pronucleus (Raja, 2005).

Mst35Ba and Mst35Bb are present at cytological position 35B6 and 35B6-7, respectively, on the chromosome arm 2L. These two genes are arranged in tandem, and both consist of three exons. The 5'UTR, coding region, and the 3'UTR of these genes are highly identical; they probably arose from a recent gene duplication. The encoded protamines show over 94% identity to each other (Raja, 2005).

A remarkable feature of protamines is their ability to form intermolecular disulfide bridges, which is reflected by the conserved cysteine residues within mammalian protamines. The dProtA and dProtB are of 146 amino acids (aa) and 144 aa, respectively, and thus longer than even the human and mouse Protamine-2, which are 102 aa and 107 aa, respectively. Both Drosophila protamines contain 10 cysteines each and show significant similarity, particularly with respect to a high cysteine, lysine, and arginine content to mammalian protamines. Human and mouse Protamine-1 aligns to the N-terminal half of the Drosophila protamines (from aa positions 27 to 82), and four cysteine residues are conserved and regularly spaced. In contrast, Protamine-2 of human and mouse shows relatively high similarity to the C-terminal half of the Drosophila protamines, with four cysteines in this region that are conserved and regularly spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).

Mst77F is present at the cytological position 77F on the chromosome arm 3L and lies within the large intron of PKA-R1. Mst77F is also male specifically transcribed, and the encoded protein has been proposed to be a linker histone H1/H5 type, which could also play the role of a transition protein or a protamine. The Mst77F protein shares a significant similarity to the HILS1 protein of mouse and human HILS1, where the percentages of cysteine, lysine, and arginine are similar to that of mHILS1 and hHILS1. HILS1 protein has been recently described as a component of the mammalian sperm nucleus. Drosophila Mst77F encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of 9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F. Mst77F contains 10 cystine residues as in Drosophila protamines, and mHILS1 contains eight cystine residues, of which four residues are conserved (Raja, 2005).

As there are considerable differences between the mammalian protamines as well as between the mammalian HILS1 proteins and the presumptive Drosophila homologue Mst77F, additional experiments are essential to clarify if these proteins are indeed involved in the condensation of sperm chromatin (Raja, 2005).

Drosophila protamine mRNAs are transcribed at the primary spermatocyte stage, whereas in mammals protamine mRNAs are synthesized at the round spermatid stage and translationally repressed until the elongated stage, which is mediated by 3'UTR. The Drosophila ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the respective protamine genes. Nevertheless, the transgenic flies carrying these constructs still show repression of translation. So, in Drosophila, the region responsible for the translational repression is most likely in the 5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to the reporter lacZ show that the translation repression element is indeed present in the 5'UTR. This holds true also for the mRNA of the Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated concerning translational repression so far in male germ lines of Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription ceases already with the entry into meiotic divisions. Since the protamines are made in the elongated spermatids, the transcriptional silencing in Drosophila spermatogenesis seems to be independent of protamines (Raja, 2005).

When primary amino acid sequences of Drosophila protamines are compared to mammalian protamines, it is quite evident that Drosophila protamines are relatively large. dProtA and dProtB are over 94% identical to each other. This could explain that both the protamines may be functionally redundant. Human and mouse Protamine-1 aligns with the N terminus of both Drosophila protamines, and Protamine-2 aligns more to the C terminus. It is possible that the Drosophila protamines undergo posttranslational cleavage at the N terminus, as is known for mammals. The cytoplasmic eGFP fused at the C terminus shows clear nuclear localization, indicating that the tagged protamine is functionally intact. Drosophila protamines each contain 10 cysteine residues at identical positions, while over 4 of 10 cysteines at the N terminus and the C terminus are conserved with human and mouse Protamine-1 and Protamine-2, respectively. With nine cysteines, the content is highest in Protamine-1 of mice. Inter- or intra-disulfide bridges can be formed between the cysteine-rich protamines to condense the DNA. For mice it is shown that mutation in protamine-1 or protamine-2 is haploinsufficient and causes male sterility. A haploid situation was analyzed for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+; these flies are fertile males and show normal spermatogenesis. The large amount of identity that both dProtA and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).

Chromatin reorganization is an essential feature during spermiogenesis. The functional significance of chromatin compaction during spermiogenesis is still unknown. The main explanation seems to be that compaction of the sperm nucleus is an essential factor for its mobility as well as for the penetration of sperm into the egg and genomic stability. In mammals, somatic histones are in part replaced by spermatid-specific variants during meiotic prophase, later by major transition proteins TP1 and TP2, and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements lead to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing major chromatin condensing proteins and DNA in the nucleus (Raja, 2005).

In Drosophila, so far no proteins have been identified that are involved in the packaging of the genome in the mature sperm nucleus. One observation, that Histone3.3 variant and the somatic H3 isoform in Drosophila are vanishing at the time of chromatin condensation, supports the view of histone displacement, but it was still a question of whether it is the real absence of histones at this stage in Drosophila or whether the antibodies are not accessible to the mature sperm due to the tight packaging of the chromatin. To circumvent this problem, the GFP fusion approach was chosen, use was made of the existing His2AvDGFP, and Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated in order to analyze the situation in Drosophila. The results clearly show that histone His2AvD is lost from the spermatid nuclei at the time of appearance of protamines and Mst77F during later stages of spermatid differentiation. The exact molecular mechanisms underlying the histone displacement, degradation, and incorporation of protamines onto the chromatin are poorly understood. For mammals, evidence has been obtained that histone H2A is ubiquitinated in mouse spermatids around the developmental time period when histones are removed from the chromatin. The mammalian HR6B ubiquitin-conjugating enzyme is the homologue of yeast RAD6, and both can ubiquitinate histones in vitro. Thus far, the mechanism of histone displacement and protamine incorporation is unknown during spermiogenesis in Drosophila. In flies as well as in mammals, many questions remain unanswered that need to be addressed about these underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).

In mammals, transition proteins act as intermediates in the histone-to-protamine transition. In mice, the onset of HILS1 and transition proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm. Mice lacking both TP1 and TP2 show normal transcriptional repression, histone displacement, nuclear shaping, and protamine deposition but show the loss of genomic integrity with large numbers of DNA breaks leading to male sterility. In Drosophila, histones are displaced with synchronous accumulation of protamines and Mst77F. Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has been proposed to play a role either as a transition protein or as a protamine for compaction of the Drosophila sperm chromatin. Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic amino acid content but not to mouse TP1, TP2, or H1t. Moreover, the results show that the pattern of expression of Mst77F in the nucleus is similar to that of mHILS1 in the nucleus, with the exception that Mst77F is also transiently detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In mammalian mature sperm nuclei, it is only the protamines that are the chromatin condensing proteins which persist. This again raises the question of whether Mst77F could also play the role of protamines. However, one additional copy of dProtB (dProtA and dProtB showing 94% identity may be functionally redundant) does not rescue the ms(3)nc3 phenotype, indicating that the role of Mst77F may be completely or partially different from that of protamines in the nucleus. However, a null mutation for Mst77F is required to answer this question with respect to chromatin condensation. In ms(3)nc3 mutants, the chromatin condensation with the native protamines continues to take place. When a closer look was taken at the deposition of ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes, it revealed that the condensed chromatin in the tid-shaped nuclei is concentrated at the two opposite ends, with a lightly stained chromatin spaced in the center. So the chromatin condensation takes place but may not be complete with the incorporation of the mutant Mst77F protein. The large amount of chromatin compaction or condensation seen in Drosophila mature sperm when compared to that of mouse and human sperm possibly could be the result of persistence of Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).

ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify interacting proteins involved in microtubule function in Drosophila. This study shows that ms(3)nc3 is a single missense mutation from a T>A transition, causing the substitution of threonine instead of serine at aa position 149. Mst77F shows a pattern of expression similar to protamines in the nucleus and was also seen in the flagella until the individualization stage. Since ms(3)nc3 fails to complement class I alleles at the ß2 tubulin locus, it is possible that Mst77F has a dual role to play as a chromatin condensing protein in the nucleus and for the normal nuclear shaping. Nuclear shaping is a microtubule-based event. ms(3)nc3 leads to a tid-shaped nuclear phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar defective nuclear shaping is seen with the few homozygous and heteroallelic combinations of class I alleles of ß2 tubulin. The incorporation of the defective subunit encoded by ms(3)nc3 may interfere with the function of the resulting complex. These data suggest the involvement of an Mst77F (a linker histone variant) in the microtubule dynamics during the nuclear shaping. This again complements the role of sea urchin histone H1 in the stabilization of flagellar microtubules (Raja, 2005).

After the first steps in the fertilization process, the male gamete is still in the highly compact protamine-based chromatin structure. In a wild-type egg, the paternal pronucleus changes the shape from the needle-like to a spherical structure. Furthermore, the male pronucleus acquires a nucleosome-based structure before zygote formation and thus is transformed into a replication-competent male pronucleus. sesame is a maternal effect mutation in HIRA and had been mapped to 7C1. HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In Drosophila, HIRA is expressed in the female germ line and a high level of HIRA mRNA is deposited in the egg. Human HIRA is shown to bind to histone H2B and H4. The WD repeats present at the N-terminal part of HIRA could probably function as a part of a multiprotein complex. Xenopus HIRA proteins are also known in promoting chromatin assembly that is independent of DNA synthesis in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which strengthens the function of HIRA in binding to the core histones. This study shows that neither Drosophila protamine is removed from the male pronucleus in sesame mutants. This leads to the proposal that the transport and incorporation of histones onto the chromatin in some manner is coupled to the removal of protamines in which HIRA could play an important role in the multiprotein complex required in this chromatin reconstitution process. Mst77F removal from the male pronucleus in contrast to protamines is independent of HIRA (Raja, 2005).

During spermiogenesis, chromatin reorganization of the complete genome is an essential feature for male fertility. This process leads to an extremely condensed state of the haploid genome in the sperm and requires a reorganization of the paternal genome in the male pronucleus during fertilization and before zygote formation. With the characterization of the chromatin condensing proteins in Drosophila, it would be possible to gain more insight into the mechanisms of sperm chromatin reorganization during spermiogenesis and fertilization (Raja, 2005).

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, a functional genetic screen was conducted of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas (CG13541), required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis (Rivard, 2021).

In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. This study focused on post-meiotic stages and shows that also after meiosis, histone H3 shows a high overall methylation of K9 and K27; it was hypothesised that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, histones are lost during the early canoe stage, and just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. It is believed that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, the first transition protein-like chromosomal protein to be found in Drosophila, Tpl94D, is reported. It is proposed that Tpl94D (an HMG box protein) and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, it is showm that histone modifications and removal are independent of protamine synthesis (Rathke, 2007).

The switch between a nucleosome-based chromatin configuration and a protamine-based structure is a specialised form of chromatin remodelling in the male germline. The mammalian zinc finger protein CTCF is involved in many epigenetic processes. Furthermore, paralogous variant of CTCF which is testis-specifically expressed, called BORIS, is exclusively expressed in the mammalian male germline. The function of BORIS in this context is still not clear. Drosophila, in contrast to mammals, contains only one CTCF gene. It was therefore asked whether Drosophila CTCF is also expressed in the testes, and immunostaining and anti-histone staining was performed on testes of transgenic flies expressing protamine-eGFP. CTCF expression was observed during pre-meiotic and meiotic stages at the chromosomes as has been shown for mitotic cell division in mammalian cell culture. Shortly after meiosis, CTCF is visible in young elongating nuclei, where it co-localises with the chromatin as indicated by the histone distribution. CTCF is also present in the early and late canoe stage spermatid heads. At the early canoe stage, CTCF is very diffusely distributed in comparison to histones. CTCF does not co-localise with the chromatin which starts to condense at one side of the nucleus. This diffuse distribution is still visible at the late canoe stage when protamine-eGFP starts to be deposited to the chromatin. CTCF is no longer detectable after the canoe stage. The earlier chromatin-associated CTCF localisation might indicate a very early role in chromatin reorganisation at the switch between the nebenkern and canoe stage. Furthermore, CTCF might be associated primarily with the chromatin, which is not yet condensing during these stages. The late canoe stage is the only post-meiotic stage where distinct regions of RNA polymerase II are found with an antibody directed against a phosphorylated subunit of active polymerase, indicative of transcription. At this precise stage, only a very small set of genes is thought to be transcribed. Also CTCF expression during chromatin reorganisation in the nucleus was detected in D. hydei (Rathke, 2007).

Sperm morphogenesis is characterised by an impressive degree of changes in cell architecture based on stored, translationally repressed mRNAs that are recruited at the appropriate time to the polysomes. Among these are mRNAs that encode Tpl^94D and protamines. A dramatic switch in structure from the nucleosomal- to the protamine-based structure of chromatin takes place, and this remarkable chromatin reorganisation of the complete genome is a typical feature depending on stored mRNAs, e.g. for protamine synthesis. This process ultimately leads to an extremely condensed state of the haploid genome in the sperm, which is essential for male fertility in mammals. This study focused on the switch between a nucleosomal- and a protamine-based chromatin reorganisation. The major steps in chromatin organisation take place in the canoe stage of spermatid development. A candidate for a transition protein in Drosophila was identified. The corresponding gene tpl^94D (CG31281) encodes a predicted basic high mobility group (HMG) protein of 18.8 kDa. In transgenic flies, Tpl^94D-eGFP fusion proteins are expressed solely during the switch between histones and protamines, as is typical for mammalian transition proteins. Since a highly similar chain of events to those reported in mammals is observed, the Drosophila system is considered an excellent choice to study the mechanism of chromatin remodelling during male germ cell development (Rathke, 2007).

Generally, the bulk of histones, including their diverse modifications in the N-terminal tail, appear to be removed during the canoe stage. Furthermore, the nucleus accumulates ubiquitin at the early canoe stage, when mono-ubiquitylation of histone H2A is no longer detectable. Therefore, taking into account the known presence of proteasomes in the nucleus at this stage of chromatin reorganisation and the overlap of expression shown in this study, it is hypothesised that this ubiquitylation is targeting histones for degradation. This study investigated several mutants having mutations in ubiquitin-conjugating enzymes or ubiquitin ligases, exhibiting arrested spermiogenesis during spermatid development and that are male sterile. However, in all investigated mutants, histone removal is indistinguishable from that of wild-type flies (Rathke, 2007).

Many histone modifications were found after meiosis and were categorised into three classes (Rathke, 2007).

1. Histone modifications that persist from pre-meiotic stages and keep the genome silent.
   
   The vast majority of the genome is transcriptionally silent in post-meiotic stages. This is accompanied by multiple histone modifications that persist from pre-meiotic stages and indicate silencing such as H3K9 and H3K27 methylation. These modifications do not change significantly during post-meiotic stages, which is in agreement with the hypothesis that these modifications predominantly play a role in maintaining transcriptional silencing. Previously, phosphorylation of histones have been analysed during spermatogenesis. Phosphorylated histone H4S1 and H3S10 are present during meiotic divisions. H3S10 phosphorylation is hardly detectable after meiosis, whereas phosphorylation of H4S1 persists until chromatin compaction starts.
   
   
2. Histone modifications that persist from pre-meiotic stages and characterise transcriptionally active chromatin.
   
   The primary spermatocyte phase is characterised by a high level of transcriptional activity of housekeeping genes. In addition, genes are transcribed that are needed for the subsequent steps in spermatogenesis, as the majority of transcription ceases once meiotic division starts. H4 acetylation and H3K4 and H4R3 methylation of histones were investigated. These histone modifications, which are indicative of transcriptional activity, persist until histone degradation.
   
   
3. Increasing or de novo appearance of histone modifications that decrease the affinity between histones and DNA as a prelude to histone removal.
   
   It might be that H4 hyper-acetylation, as postulated for mammals and/or other secondary modifications of histones are the first step towards histone removal. The fact that these modifications are conserved between mammals and flies adds support to this hypothesis. Indeed, histone H4 acetylation is very pronounced at the canoe stage and de novo mono-ubiquitylation of histone H2A is seen in round spermatids. Both types of histone modifications are proposed to be necessary for opening the chromatin and decreasing the contact between DNA and histones. The fact that histone H2A mono-ubiquitylation vanishes before the early canoe stage, thus before the hyper-acetylation of histone H4, leads to thinking about a stepwise remodelling of the chromatin. This study proposes that these histone modifications open the chromatin, so that enzymes and regulators have access to histone-based chromatin and can induce and prepare the reorganisation of the genome in the male germline.
   

It remains to be clarified whether and how these histone modifications influence the topology of the chromatin as a prelude to histone removal as well as for Tpl^94D, Mst77F and protamine deposition. A functional approach based on analysis of mutants of histone-modifying enzymes is difficult, as all characterised histone-modifying enzymes are already active during Drosophila development or at least in spermatogonia and spermatocytes. Therefore a tissue-specific knock-out mutant would most probably exhibit arrest of spermatogenesis before meiosis, rendering it useless for experimental purposes (Rathke, 2007).

At the first glance, it might seem surprising that histones and all their modifications are removed. Instead of specifically reverting the differentially modified histones to their unmodified state, they are removed together with all histones. This might allow the paternal genome to form nucleosomes with unmodified histones after fertilisation and before zygote formation. Thus, the paternal genome starts embryogenesis with a nucleosomal chromatin lacking histone modifications (Rathke, 2007).

The data show that most of the histones are removed between the early and late canoe stage; such a process requires a loosening of contact between the histones and DNA, which in turn requires an unwinding of the chromatin structure. It is proposed that this unwinding process is facilitated by DNA nicks as they were widespread at this stage of chromatin reorganisation. Finally, Tpl^94D, UbcD6 and SUMO were also observed to accumulate in the chromatin during this process. DNA breaks, Tpl^94D, UbcD6 and SUMO were no longer detectable when protamines were fully expressed. Thus, it is proposed that all these proteins and the DNA breaks act together in an unknown manner to allow chromatin remodelling (Rathke, 2007).

The CTCF protein is present during pre-meiotic stages in the nucleus and stays associated with the chromosomes during meiosis. After meiosis, however, strong localisation to the nucleus is detected during the transition from round spermatid nuclei to the early canoe stage of spermiogenesis. It is speculated that CTCF might set borders in the chromatin for the histone modifications, which are characteristic of the canoe stage, such as acetylation and ubiquitylation. CTCF is visible for longer than histones and disappears together with active RNA polymerase II. CTCF might maintain chromatin accessibility to RNA polymerase II since a few genes are known to be transcribed at this time. In addition, transient occurrence of RNA polymerase II at the late canoe stage might require CTCF to insulate active genes from inactive ones. This idea needs to be tested in tissue-specific CTCF loss-of-function mutants; such mutants are, however, currently unavailable (Rathke, 2007).

The question of whether histone removal is dependent on a signal that monitors the start of protamine and Mst77F mRNA translation was addressed. Both histone modification and degradation are indistinguishable from the wild-type in loss-of-function mutants of Mst35Ba and Mst35Bb, the genes encoding protamine A and B, respectively. Also in nc3 mutants of Mst77F, histone removal is not disturbed. It is concluded that N-terminal tail modification of histones and histone degradation, on the one hand, and protamine deposition, on the other, are controlled by different pathways in the cell (Rathke, 2007).

In mammals, it is well known that after meiosis the nucleosomal conformation is lost. This is accompanied by the appearance of testis-specific linker histones. So far, no linker histone variants have been identified in Drosophila, but variants of H2A (H2AvD) and H3 (H3.3) are known. In mammals, histones are hyper-acetylated before being displaced from the DNA, and phosphorylation and ubiquitylation have also been proposed to occur. For Drosophila, H2A mono-ubiquitylation and a strong increase in H4 acetylation occur shortly before histone removal and degradation. In mammals, histones are replaced first by transition proteins (major types: TP1 and TP2). This study identified the high mobility group protein Tpl^94D, a first probable candidate for a functional homologue of mammalian transition proteins. In mammals, transition proteins are subsequently replaced by protamines leading to chromatin with a doughnut structure. In Drosophila, it has recently been shown that the sperm nucleus also contains protamines. Protamines A and B are encoded by two closely related protamine genes, Mst35Ba and Mst35Bb. In addition, the identification of Mst77F shows that sperm nuclei contain at least one further abundant chromatin component. Moreover, in human sperm several new putative protamines have been identified by 2D gel electrophoresis and protein sequencing. In mammals, this chromatin reorganisation is essential for male fertility. Male flies carrying the deletion protDelta38.1, where both protamines as well as three additional ORFs are removed, show severely reduced fertility (Rathke, 2007).

In summary, a step-by-step scheme is proposed for chromatin reorganisation: (1) histone modifications lead to subsequent histone removal and degradation; (2) the exposed chromatin becomes nicked, resulting in DNA breaks; (3) Tpl^94D deposition constitutes an intermediate stage that triggers subsequent protamine-based chromatin organisation (Rathke, 2007).

Since many features concerning spermiogenesis are conserved between Drosophila and mammals, it is proposed that Drosophila is an ideal system to gain further insight into the mechanism of chromatin reorganisation in spermatid nuclei, a process that is crucial for male fertility (Rathke, 2007).

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning, and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP, and EGF pathways are used to mediate germline-soma communication. This study demonstrates that gap junctions may also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg/Inx4 and Inx2 on the germline and the soma side, respectively. Overall, the results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, this study shows that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues (Smendziuk, 2015).

The work presented in this study demonstrates that gap junctions between the soma and germline are essential for fly spermatogenesis. Previous work showing an essential role for Zpg in the fly gonads raised the possibility that signals either from the soma or from other germ cells travel through gap junctions to regulate germline survival and differentiation. Subsequent work in the fly ovaries showed that Zpg was also required for GSC maintenance. This analysis supports and extends these conclusions by finding a cell-autonomous requirement for Zpg in GSC maintenance in the fly testis and demonstrates a role for Inx2 in the soma. Furthermore, it was found that gap junction-mediated signals from the germline also play unique and essential roles in the soma during spermatogenesis, independent of general germline defects. In particular, gap junctions are required to control the proliferation of CySCs and promote the differentiation of their daughters. This work illustrates that the main type of gap junction between the soma and the germline in the fly testis is a heterotypic channel coupling Inx2 in the soma and Zpg in the germline. Importantly, disrupting gap junctions in the soma by knocking down Inx2 phenocopies the zpg mutant phenotype in both the germline and soma. Therefore gap junction-mediated soma-germline regulation in the testis is bi-directional (Smendziuk, 2015).

Gap junctions contribute to stem cell regulation in the testis Recent work has highlighted the importance of gap junctions in stem cell regulation in a number of systems. In line with results from other stem cell models, the data illustrates a specific role for gap junctions in both GSCs and CySCs. The role of gap junctions in stem cell regulation in the testes was illustrated by the requirement for Zpg in the germline and Inx2 in the soma for GSC maintenance. Moreover, loss of Zpg or somatic knockdown of Inx2 also affected CySC proliferation. Furthermore, ultrastructural analysis revealed the presence of gap junctions between GSCs and CySCs. These results, as a whole, suggest a requirement for gap junction-mediated soma-germline communication in both stem cell populations and at the earliest stages of sperm differentiation (Smendziuk, 2015).

Gap junctions facilitate signalling between the soma and germline Following the stem cell stage, strong expression and co-localization of Zpg and Inx2 was consistently detected starting at the 4-cell cyst stage. Expression of Zpg and Inx2 began to diminish after the early spermatocyte stages and was not detected past meiotic stages. The timing at which Inx2 and Zpg expression were most prominent corresponds to a period during which niche signals such as BMP are lost. Loss of these signals causes the germline to undergo rapid differentiation and specialization. It has been shown that as somatic cells move away from the niche and begin differentiating, the soma forms a permeability barrier around the germline, isolating the germline from the outside environment. This transition corresponds with a switch occurs whereby soma-germline communication shifts from predominantly exocrine to juxtacrine signalling. Thus, as the germline becomes increasingly isolated, it becomes more dependent on differentiation signals that arrive via gap junctions from the soma. Once the germline becomes isolated, gap junctions may also play an important nutritive role and permit the movement of essential small metabolites between the germline and soma. Similarly, the soma requires gap junction-mediated signals to allow it to accommodate the increasingly expanded, differentiated, and specialized germline (Smendziuk, 2015).

The observations that gap junctions regulate germline differentiation and soma proliferation are in line with studies from both vertebrate and invertebrate models. In C. elegans, it was recently shown that gap junction-mediated signals are required to maintain GSCs in the niche and for germline differentiation (Starich, 2014). Similarly, work in vertebrates has shown that loss of gap junction-mediated signalling in the soma increased proliferation in post-mitotic Sertoli cells. It is therefore likely that an early role for gap junctions in coordinating soma-germline differentiation is an evolutionarily-conserved mechanism. One recurring feature of germline-soma gap junctions is the expression of different gap junction proteins, resulting in heterotypic gap junctions, exemplified by the Inx2-Zpg gap junctions observed in flies. A key problem in understanding the role of gap junctions in mediating soma-germline communication is identifying the transported signalling cargos. Some possible signals are cAMP, Ca2+, and cGMP, which have been implicated in regulating meiosis in the germline. Attempts to study cAMP and Ca2+ in the testis have proven inconclusive. However, recent work in Drosophila ovaries has suggested that somatic gap junctions may play roles in regulating pH, membrane potential, and ion transport. Overall, multiple signals are likely exchanged between the soma and germline through gap junctions and elucidating their respective functions is a complex task that should be further studied (Smendziuk, 2015).

Based on the results presented in this study, the following model is proposed: GSCs receive multiple cues that control their behaviour, with gap junctions mostly provide a supporting role, allowing the passage of cues from the soma that facilitate long-term GSC maintenance. After stem cell division germline undergoes rapid differentiation. The germline becomes increasingly isolated from the outside environment, and a permeability barrier is formed by the soma. As outside signals from the niche are lost, the germline relies more heavily on gap junctions to allow the passage of small molecules and metabolites from the soma to promote differentiation and provide nourishment. To ensure coordinated growth and differentiation of the soma and germline, signals pass from the germline through the gap junctions into the soma. Taken together, this work defines gap junction-mediated juxtacrine signalling as an additional signalling mechanism in the fly testis. Furthermore, this study provides a clear illustration of the bi-directional regulatory action of soma-germline gap junctions. As this study demonstrates, disrupting innexins in the soma or germline leads to a specific regulatory effect in the other tissue. Therefore bi-directional gap junction-mediated signalling plays a vital role in ensuring proper coordination of the soma and germline during spermatogenesis (Smendziuk, 2015).

In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. This study shows that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35a/Mst35b. Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals (Kimura, 2016).

In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. This study shows that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization (Tirmarche, 2016).

In sexually reproducing animals, the differentiation of haploid spermatids into mature spermatozoa involves major reorganization of the nuclear architecture1. Starting as round nuclei after the second male meiotic division, spermatid nuclei slowly transform to eventually acquire the final, species-specific shape of mature sperm nuclei. Extreme compaction of nuclear DNA generally accompanies the streamlining of spermatids, resulting in the shutdown of basic nuclear activities, including transcription and DNA repair. In most species, sperm nuclear compaction requires the massive replacement of somatic-type histones with sperm nuclear basic proteins (SNBPs). SNBPs encompass a heterogeneous group of chromosomal proteins that are specifically expressed in male germ cells and deposited during spermiogenesis. Protamines, the best characterized SNBPs, are small (50-60 aa), arginine rich proteins present in vertebrates and some invertebrates. A model based on mammalian protamines has proposed that these positively charged proteins bind the major groove of the double helix and form toroid-like structures containing about 50 kilobases of sperm DNA. Protamines in eutherian mammals and a few other animal groups are also enriched in cysteine residues, which are otherwise rare in chromosomal proteins, including histones and most SNBPs. During sperm maturation in eutherian mammals, oxidation of the protamine cysteine thiols (-SH) allows the formation of a tridimensional network of disulfide bridges (-S-S-). Intermolecular disulfide crosslinks notably participate in the stabilization of sperm chromatin by connecting adjacent chromatin fibres. It is actually well established that for most species of mammals, a thiol reducing agent, such as dithiothreitol (DTT), is required to elicit sperm nuclear decondensation in vitro. In addition, it has been alternatively proposed that in human sperm, protamines thiols are non-covalently bridged by Zinc (Zn2+), thereby preventing or limiting the formation of excess disulfide bonds that could perturb sperm nuclear decondensation at fertilization (Tirmarche, 2016).

This work functionally characterized the Drosophila maternal thioredoxin Deadhead (DHD) and we demonstrate that DHD is required to unlock sperm chromatin at fertilization. Thioredoxins are small redox proteins found in all organisms. They typically reduce disulfide bonds on target proteins using a pair of cysteine thiols present in their conserved active CGPC site. Thioredoxins play important metabolic, protective or signalling functions but the molecular bases of their target specificity or functional specialization remain poorly understood. Three classical thioredoxins are found in Drosophila melanogaster. Trx-2 is a non-essential ubiquitous protein that participates in the protection against oxidative stress, whereas TrxT and DHD are sex-specific thioredoxins encoded by a pair of adjacent genes. While the role of the testis-specific TrxT is unknown, DHD is specifically expressed in the female germline and is essential for embryo development19,20. In this work, we demonstrate that DHD is essential for sperm nuclear decondensation at fertilization (Tirmarche, 2016).

The stabilization of sperm chromatin with disulfide bonds is a remarkable innovation that emerged independently in distantly-related animal groups with the selective acquisition of cysteine residues in SNBPs. This work first established that Drosophila sperm chromatin, which is packaged with cysteine-rich SNBPs, is stabilized by disulfide crosslinks. The reducing agent DTT allows efficient decompaction of Drosophila sperm nuclei in vitro, in a way similar to its well-established activity on sperm nuclei from eutherian mammals, for instance. Similarly, it has been recently shown that pre-treatment of Drosophila sperm nuclei with DTT improves antibody accessibility to nuclear epitopes in immunofluorescence experiments. Although little is known about the structural organization of Drosophila sperm chromatin, a recent study showed that the Drosophila SNBP Mst77F is a DNA binding protein which induces DNA condensation in vitro through a multimerization process involving its coiled-coil domain. Oxidation of cysteine thiols present in Mst77F could participate in the stabilization of this unique chromatin architecture through the establishment of disulfide crosslinks. Disulfide bonds could also allow the formation of inter-chromatin fibre associations, as proposed for mammalian sperm chromatin (Tirmarche, 2016).

The implication of DHD in the reduction of sperm chromatin disulfide crosslinks in Drosophila eggs is first supported by a detailed characterization of the dhd phenotype. Among the rare Drosophila maternal effect mutants affecting sperm chromatin remodelling, dhd is unique and shows the earliest phenotype reported so far. Notably, in dhd mutant eggs, the sperm nucleus is initially intact, showing no sign of decondensation. Second, any role of DHD in the reduction of sperm nuclear disulfide bonds implies the critical requirement of its catalytic redox motif. Previous work has demonstrated that transgenes expressing DHD in which the two active cysteines are replaced by serines failed to rescue the fertility of mutant females. This work has shown that replacing the second cysteine residue of the redox motif not only abolished DHD function but also trapped the protein on sperm chromatin in a remarkably specific manner. This experiment strongly suggests that DHD directly targets disulfide bonds on sperm chromatin. Interestingly, the DHDC34S mutant protein could not rescue the dhd nuclear phenotype despite its expected ability to cleave its disulfide targets. It is speculated that the irreversible covalent trapping of DHDC34S on SNBPs at the surface of sperm chromatin could impede their eviction and prevent further access of the thioredoxin to the compact sperm nucleus. It is proposed that, following sperm penetration through the egg micropyle and sperm plasma membrane breakdown, the sperm nucleus is exposed to the egg cytoplasm that contains massive amounts of DHD thioredoxin. The reduction of disulfide crosslinks by DHD induces sperm nuclear decompaction and allows the subsequent extraction of SNBPs from sperm chromatin, a process that likely involves the ISWI chromatin remodeler. Finally, the removal of SNBP leaves DNA accessible to the HIRA complex for genome-wide nucleosome assembly with maternally supplied histones (Tirmarche, 2016).

In mammals, the tripeptide antioxidant glutathione is abundant in oocytes and plays an important role in sperm nuclear decondensation. Inhibition of glutathione synthesis by buthionine sulfoximine during oocyte maturation indeed efficiently blocks sperm nuclear decondensation and this effect can be reversed by DTT. Whether glutathione directly reduces SNBP disulfides in vivo or instead activates a dedicated disulfide reductase is still unresolved. In most species, glutathione disulfide (GSSG) is reduced into glutathione (GSH) by the highly conserved glutathione reductase enzyme. Drosophila is unusual in lacking glutathione reductase and instead relies on the thioredoxin system for GSSG reduction. Interestingly, biochemical characterization of Drosophila thioredoxins have established that DHD and Trx-2 are equally efficient for GSSG reduction in vitro, opening the possibility that DHD could indirectly influence sperm nuclear decondensation by controlling the level of GSH in eggs. However, the demonstration that recombinant DHD protein is sufficient for sperm nuclear decondensation and SNBP removal in an in vitro assay argues against this hypothesis. Still, this study has clearly documented that sperm chromatin remodelling is not completely blocked in dhd eggs but instead occurs very slowly. This slow replacement of SNBPs with histones suggests that mutant eggs still have residual reducing power for the cleavage of SNBP disulfide bonds, an effect that could likely involve glutathione itself (Tirmarche, 2016).

This study also revealed that the migration of the female pronucleus to the centre of the egg fails in a majority of dhd eggs, thus preventing the initiation of embryo development. The release of paternal centrioles from the base of the sperm nucleus and the subsequent elaboration of the sperm aster were indeed affected in mutant eggs. As a matter of fact, depletion of glutathione in bovine oocytes not only blocks sperm nuclear decondensation but also prevents sperm aster growth and pronuclear migration. It was observed that the disassembly of the sperm tail and connecting piece, as well as the release of the proximal centriole were frequently affected in treated oocytes, suggesting that the role of glutathione at fertilization is not restricted to sperm chromatin decompaction. Further investigation will be required to determine whether DHD similarly targets additional sperm structures (Tirmarche, 2016).

In conclusion, it is proposed that the rapid unlocking of sperm chromatin at fertilization in Drosophila is critically dependent on the redox activity of the egg specific DHD thioredoxin. dhd is apparently the most recent thioredoxin gene in Drosophila and likely originated after the duplication of the ancestral Trx-2 gene. It is thus tempting to propose that the emergence of this highly specialized protein could reflect the adaptation of the oocyte to the rapid evolution of sperm chromatin architecture. Future work will aim at understanding how the structural features of DHD underlie its crucial specialization at the onset of embryo development (Tirmarche, 2016).

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. This study shows that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, a reduction in PI31 levels was observed in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. These results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice (Rathje, 2019).

Autophagy is a conserved, lysosome-dependent catabolic process of eukaryotic cells which is involved in cellular differentiation. Its specific role in the differentiation of spermatogonial cells in the Drosophila testis was studied. In the apical part of the Drosophila testis, there is a niche of germline stem cells (GSCs), which are connected to hub cells. Hub cells emit a ligand for bone morhphogenetic protein (BMP)-mediated signalling that represses Bam (bag of marbles) expression in GSCs to maintain them in an undifferentiated state. GSCs divide asymmetrically, and one of the daughter cells differentiates into a gonialblast, which eventually generates a cluster of spermatogonia (SG) by mitoses. Bam is active in SG, and defects in Bam function arrest these cells at mitosis. This study shows that BMP signalling represses autophagy in GSCs, but upregulates the process in SG. Inhibiting autophagy in SG results in an overproliferating phenotype similar to that caused by bam mutations. Furthermore, Bam deficiency leads to a failure in downstream mechanisms of the autophagic breakdown. These results suggest that the BMP-Bam signalling axis regulates developmental autophagy in the Drosophila testis, and that acidic breakdown of cellular materials is required for spermatogonial differentiation (Vargo, 2022).

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study shows that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase (Baker, 2023).

DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. By using in vitro biochemical assays this study identified chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. This study has identified IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. In addition to recognized roles in protein traffic, a nuclear transport receptor can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone (Emelyanov, 2023).

During spermatogenesis, mitochondria extend along the whole length of spermatid tail and offer a structural platform for microtubule reorganization and synchronized spermatid individualization, that eventually helps to generate mature sperm in Drosophila. However, the regulatory mechanism of spermatid mitochondria during elongation remains largely unknown. This study demonstrated that NADH dehydrogenase (ubiquinone) 42 kDa subunit (ND-42) was essential for male fertility and spermatid elongation in Drosophila. Moreover, ND-42 depletion led to mitochondrial disorders in Drosophila testes. Based on single-cell RNA-sequencing (scRNA-seq), this study identified 15 distinct cell clusters, including several unanticipated transitional subpopulations or differentiative stages for testicular germ cell complexity in Drosophila testes. Enrichments of the transcriptional regulatory network in the late-stage cell populations revealed key roles of ND-42 in mitochondria and its related biological processes during spermatid elongation. Notably, it was demonstrated that ND-42 depletion led to maintenance defects of the major mitochondrial derivative and the minor mitochondrial derivative by affecting mitochondrial membrane potential and mitochondrial-encoded genes. This study proposes a novel regulatory mechanism of ND-42 for spermatid mitochondrial derivative maintenance, contributing to a better understanding of spermatid elongation (Yu, 2023).

Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the Transition-protein-like 94D (Tpl94D) contains an HMG-box domain and is expressed during chromatin reorganization. This study searched for additional HMG-box-containing proteins with a similar expression pattern. Two proteins specifically expressed in the testis, tHMG-1 (CG12104) and tHMG-2 (CG30356), whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. Tpl94D, tHMG-1 and tHMG-2 were shown to localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. These results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. It is hypothesized that the mechanism of chromatin reorganization is a process highly secured by redundancies (Gartner, 2014).

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Using CRISPR/Cas9 mutagenesis, this study determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, the data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. These results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis (Shao, 2023).

The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. This study identifies Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin (Eren-Ghiani, 2015).

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. This study shows that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs, leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. It was further shown that modulo regulates these protamine genes at the step of transcript polyadenylation. It is concluded that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility (Park, 2023).

The present study reveals a regulatory mechanism mediated by a nucleolar protein Modulo that balances the expression of protamine subtypes in D. melanogaster. This finding may represent a similar theme to what is seen in the fragile balance of PRM1 and PRM2 in mammalian fertility. In the case of Mst77Y, Y-linked multicopy Mst77F homologs, this study suggests that they have the ability to act as dominant-negative protamines and thus must be carefully regulated/repressed. The present study also confirmed that Mst77Y genes are expressed as proteins as suggested previously by the finding that several of the copies contain complete open-reading frames and is also consistent with small RNA sequencing revealing that the Mst77Y locus is not a source of small RNAs (Park, 2023).

Overexpression of Mst77Y dominantly interferes with Mst77F incorporation, leading to decompaction of sperm nuclei and their demise. Mst77Y genes feature differences from their autosomal homolog that further support the idea that they are dominant-negative versions of Mst77F and interfere with sperm chromatin compaction. Mst77Y-12, which retains the full ORF of Mst77F, exhibits 87% overall sequence homology to autosomal Mst77F. At the domain/motif level, the MST-HMG-box domain, suggested to be important for DNA binding, maintains 100% homology, while the coiled-coil motif and C-terminal domain maintain only ~79.5% and ~85% homology, respectively. It has been shown that the N-terminal domain of Mst77F, which contains the coiled-coil motif, interacts with the C-terminal domain to induce multimerization to mediate DNA compaction. The changes to Mst77Y at important regions may thus influence the multimerization of protamines and the formation of proper sperm chromatin structure, by interfering with the ability of the canonical version to multimerize. The notion that Mst77Y behaves as a dominant-negative version of Mst77F is further supported by the fact that overexpression of Mst77Y-3, a truncated version which does not contain the C-terminal domain, is still sufficient to cause defects in nuclear compaction (Park, 2023).

What is the potential 'function' of dominant-negative protamines? A few nonmutually exclusive possibilities are proposed. First, dominant-negative protamines may participate in meiotic drive, as suggested by recent works in D. simulans as well as D. melanogaster. Indeed, the data suggest that Mst77Y has the ability to disproportionally affect X-bearing spermatids. While this did not result in a large sex ratio distortion in offspring, this ability to harm a subset of developing spermatids during postmeiotic development may indicate the possibility that these protamine variants could be exploited by a meiotic drive system to unleash its own selfish purpose. Intriguingly, studies on the Winters sex-ratio meiotic drive system in D. simulans revealed that the driver, Dox, contains a large portion of the Protamine gene. While it has not been confirmed whether this protamine-like region is essential for sex ratio distortion, the derepression of Dox does seem to cause nuclear defects during spermiogenesis. It is proposed that a drive system that would be able to localize a dominant-negative protamine such as Mst77Y to a subset of spermatids containing one homolog over another could be quite successful at achieving bias. Alternatively, the dominant-negative version of a protamine may be utilized when spermatogenesis needs to be aborted (similar to the concept of 'programmed cell death'), for example under stress conditions. In such a case, dominant-negative protamines (such as Mst77Y) can be up-regulated to lead to abortive spermatogenesis. In such a scenario, a dominant-negative protamine may have a beneficial function for the organism. Yet another possibility that may contribute toward the rapid divergence of protamines is that protamine genes evolve to optimally package the genome, which may be greatly influenced by the composition of the most abundant sequences in a given genome, i.e., repetitive DNA such as satellite DNA. As these repetitive sequences are known to rapidly diverge across species, protamine genes may have to adapt to accommodate diverged repetitive DNA sequences, leading to rapid divergence and/or emergence of multiple protamine genes to optimally package different repetitive DNA with distinct structure/sequence. In such a scenario, fine-tuning the expression of different protamine genes may be critical. Additionally, if any protamine genes have evolved to optimally package certain satellite DNA, conversion of such protamine into a dominant-negative version can immediately target the chromosome that harbors the given satellite DNA, leading to meiotic drive that selectively harms the specific chromosome. This possibility is intriguing as the Segregation Distorter (SD) meiotic drive system in D. melanogaster is known to target Responder satellite DNA repeats and exhibits sperm nuclei decompaction similar to what is observed in this study. The possibility that dominant-negative protamines are involved in the decompaction of spermatid nuclei in the SD drive system remains to be studied (Park, 2023).

Taken together, this study identified a mechanism by which various protamine variants are coordinately regulated at the posttranscriptional level, possibly to achieve balanced expression of multiple protamine variants. A similar mechanism may be at play to fine-tune the expression levels of protamine variants in human and mouse, disruption of which is associated with compromised fertility (Park, 2023).

Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, this study comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). 6,171 proteins were identified; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate mass spectrometric data, the stage-specific synthesis and importance for male fertility were analyzed of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. This catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis (Gartner, 2019).

As ecdysteroids are produced in the reproductive organs of adult insects, and in some species influences spermatogenesis, release of vitellogenin, memory and sex-specific physiology and behavior, they share several features with the vertebrate sex-hormones. In vertebrates, the reproductive system of males primarily synthesizes testosterone, the precursor of the female sex steroid, estradiol. The conversion of testosterone to estradiol is catalyzed by a P450 enzyme, like Shd, that mediates the conversion of E to 20E in insects. Considering that E and 20E have distinct functions and have been proposed to function as sex steroids in insects, the present study provides the first evidence that males may synthesize E for sex-specific purposes whereas females presumably produce 20E (Hentze, 2013).

The secondary cells of the accessory gland require BMP signaling to regulate the synthesis or function of one or more important components of the seminal fluid as flies age and mate. However, this signaling simultaneously drives cell loss and changes in the morphology and function of the epithelium, which appears to lack regenerative capacity in flies. The prostate gland of most human males over 50 y of age is hyperplastic, and it is tempting to speculate that this reflects a regenerative response to similar events in this organ. A more detailed analysis of secondary cell biology should help to further elucidate the processes that underlie functional changes in the accessory gland epithelium and test whether these are shared by male reproductive glands in other organisms (Leiblich, 2012).

Other evidence strongly supports the idea that CD63-positive puncta secreted from SCs are exosomes and not vesicles shed from the plasma membrane. This includes the observation that CD63-positive puncta are found inside both acidic Rab7-positive MVB-like compartments as well as nonacidic Rab11-positive vacuoles and require the ESCRT and ESCRT-associated proteins Hrs and

Fucose and α-l-fucosidases have fundamental function(s) during gamete interactions. An α-l-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven Drosophila species. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-l-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. The results showed that α-l-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. No binding was detected when α-l-fucosidase was pre-incubated with fucoidan, a polymer of α-l-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-l-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila (Intra, 2015).

Insects use a spectacular variety of chemical signals to guide their social behaviours. How such chemical diversity arises is a long-standing problem in evolutionary biology. This study describes the contribution of the fatty acid elongase Bond to both pheromone diversity and male fertility in Drosophila. Genetic manipulation and mass spectrometry analysis reveal that the loss of bond eliminates the male sex pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Unexpectedly, silencing bond expression severely suppresses male fertility and the fertility of conspecific rivals. These deficits are rescued on ectopic expression of bond in the male reproductive system. A comparative analysis across six Drosophila species shows that the gain of a novel transcription initiation site is correlated with bond expression in the ejaculatory bulb, a primary site of male pheromone production. Taken together, these results indicate that modification of cis-regulatory elements and subsequent changes in gene expression pattern is one mechanism by which pheromone diversity arises (Ng, 2015)

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprises behavioral and physiological changes that include increased egg-laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP), and are maintained by SP's binding to, and slow-release from, sperm. The accessory gland contains two secretory cell types whose morphology and development differs. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, this study analyzed iab6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, 77 genes were identified whose expression is down-regulated by a factor of >5x in iab6^cocu males. By functional assays and microscopy, 20 candidate genes were tested and it was found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg-laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR (Sitnik, 2016).

PIWI-interacting RNAs (piRNAs) are predominantly produced in animal gonads to suppress transposons during germline development. Understanding about the piRNA biogenesis and function is predominantly from studies of the Drosophila female germline. piRNA pathway function in the male germline, however, remains poorly understood. To study overall and stage-specific features of piRNAs during spermatogenesis, this study analyzed small RNAs extracted from entire wild-type testes and stage-specific arrest mutant testes enriched with spermatogonia or primary spermatocytes. It was shown that most active piRNA clusters in the female germline do not majorly contribute to piRNAs in testes, and abundance patterns of piRNAs mapping to different transposon families also differ between male and female germlines. piRNA production is regulated in a stage-specific manner during spermatogenesis. The piRNAs in spermatogonia-enriched testes are predominantly transposon-mapping piRNAs, and almost half of those exhibit a ping-pong signature. In contrast, the primary spermatocyte-enriched testes have a dramatically high amount of piRNAs targeting repeats like suppressor of stellate and AT-chX The transposon-mapping piRNAs in the primary spermatocyte stages lacking Argonaute3 expression also show a ping-pong signature, albeit to a lesser extent. Consistently, argonaute3 mutant testes also retain ping-pong signature-bearing piRNAs, suggesting that a noncanonical ping-pong cycle might act during spermatogenesis. The study found stage-specific regulation of piRNA biogenesis during spermatogenesis: An active ping-pong cycle produces abundant transposon-mapping piRNAs in spermatogonia, while in primary spermatocytes, piRNAs act to suppress the repeats and transposons (Quénerch'du, 2016).

The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. This study describes the identification and initial characterization of dany (CG30401), a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC (a testis-specific meiotic arrest complex (see Always early)) and tTAFs (see Cannonball), the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, it is proposed that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization (Trost, 2016).

Meiotic sex has a deep evolutionary origin in basal eukaryotes. While meiosis has generally remained well conserved, most other aspects of sexual reproduction have diverged rapidly. In animals that differentiate a male and a female gender, male-biased genes and in particular those expressed in the germline are affected by turnover and sequence divergence that is significantly faster than in other gene classes. Analyses in Drosophila melanogaster spermatocytes have provided some of the most striking evidence for rapid evolutionary dynamics even in key elements of transcriptional networks with numerous interactions (Trost, 2016).

The comparison of different tissues in adult D. melanogaster has clearly revealed that the number of genes with an expression apparently restricted to a single tissue is maximal in the case of testis. The large majority of these, as well as of the testis-biased genes, are transcribed in spermatocytes, i.e., in germline cells during a growth phase between the last mitotic division and the onset of the meiotic divisions and spermiogenesis. The transcription of more than 1000 of these genes depends on the function of the testis meiotic arrest complex (tMAC). tMAC is a testis-specific variant of the MIP/dREAM/SynMuvB complex, a widely conserved somatic transcriptional regulator. tMAC contains subunits encoded by testis-specific paralogs (aly, tomb) that are only present within the genus Drosophila. They were identified based on a characteristic loss-of-function phenotype in which the testes fill up with spermatocytes failing to enter meiotic divisions and postmeiotic differentiation. Several genes of comparably recent origin and with similar mutant phenotypes (topi, comr, achi, vis) encode proteins interacting with tMAC. A second protein complex of paramount importance for spermatocyte-specific transcription is formed by testis-specific TATA-binding protein (TBP)-associated factors (tTAFs) in the genus Drosophila. The tTAF genes [can, mia, nht, rye (Taf12L - FlyBase), sa] were also identified based on their mutant phenotype, which is slightly milder than that associated with tMAC loss (Trost, 2016).

This study describes the identification of dany, yet another gene with a recent evolutionary origin and an essential role in spermatocyte-specific gene expression. dany is most similar to the Drosophila genes distal antenna (dan) and distal antenna-related (danr). Dan and Danr have recently been implicated in the control of neuroblast competence in Drosophila embryos, where they inhibit the repositioning of crucial target genes into repressive chromatin associated with the nuclear lamina (Kohwi, et al., 2013). Similarly, Dany is required not only for cell type-specific gene expression in spermatocytes but also for normal association of chromatin with the nuclear envelope (Trost, 2016).

This identification and characterization of dany has uncovered a factor crucial for the realization of the spermatocyte-specific gene expression program in Drosophila. Loss of dany compromises the transcriptional activation of a large number of spermatocyte-specific genes and derepresses genes that are normally inactive in spermatocytes. Moreover, dany is required for normal association of spermatocyte chromatin with the nuclear periphery (Trost, 2016).

With regard to transcriptional activation, Dany is similar to tTAFs and tMAC. Mutations in tMAC genes have a severe effect on spermatocyte-specific gene. In the case of tTAF mutants, fewer genes are affected and the reduction in transcript levels is often less severe. Loss of dany has even slightly milder effects, but there are still ~1000 genes with significantly reduced transcript levels. Many of these genes are also dependent on tMAC and tTAFs (Trost, 2016).

The fact that Dany is just as important for silencing of non-spermatocyte genes as for activation of spermatocyte-specific genes indicates that this protein does not function like the activators tTAFs and tMAC, which have not been implicated in gene silencing. Several additional observations support this conclusion. Dany intracellular localization in early spermatocytes is distinct from that of the tTAF Sa. dany transcript and protein levels depend neither on Sa nor on the tMAC components Aly and Topi. Vice versa, mutations in dany affect neither transcript levels of tTAF and tMAC genes nor accumulation and intracellular localization of representative protein products (Sa and Can) (Trost, 2016).

dany orthologs cannot be detected outside the genus Drosophila, whereas the primordial dan/danr genes are present throughout the insect lineage. The testis-specific tMAC and tTAF genes are comparably young in evolutionary terms. The fact that several functionally independent factors of paramount importance for the highly complex spermatocyte-specific gene expression program have a recent origin provides further testimony of the surprising evolutionary dynamics of genes that function in the male germline. After a gene duplication event, dany has evolved to control the expression of thousands of genes in the male germline. Dany is a potent regulator; ectopic expression of dany in somatic tissues is highly toxic (Trost, 2016).

How does Dany exert its function? Dany is a nuclear protein. After ectopic expression in larval salivary glands, it binds preferentially to all euchromatic interband regions of polytene chromosomes. In maturing spermatocytes, where it is normally expressed, Dany also appears to associate preferentially with euchromatic regions within chromosome territories and not with the regions of maximal DNA staining intensity corresponding to pericentromeric heterochromatin. As the Psq motif of some other proteins, including CENPB and transposases, is involved in sequence-specific DNA binding, it is conceivable that Dany and its closest relatives Dan and Danr bind to DNA as well. The Psq domain structure of Dan as revealed by NMR is highly similar to that observed by X-ray crystallography in CENPB. However, the DNA-binding region within CENPB is not restricted to the Psq motif but includes an additional helix-turn-helix domain that is not present in Dan, Danr and Dany. Similarly, the DNA-binding region of transposases extends considerably beyond the Psq motif. By mutating three amino acid codons within the Dany Psq motif, which correspond to positions contacting bound DNA in the case of CENPB, it was possible to demonstrate the functional importance of this motif. However, Dany^AAA still retains some function; it promotes spermatogenesis beyond the stage when dany null mutant spermatocytes arrest. Moreover, the intracellular localization of Dany^AAA in spermatocytes appears to be normal. Additional analyses will be required to resolve whether the Psq motif of Dany indeed binds DNA. It is pointed out that the Psq motif in CENPB contacts only four base pairs. Such a limited DNA sequence specificity could not explain the observed preferential association of Dany with euchromatic regions. Additional factors will have to be identified (Trost, 2016).

Interestingly, the first phenotypic abnormalities that this study has detected in dany mutants occur in young spermatocytes soon after the onset of dany expression. Whereas chromosome territories tend to become enwrapped in the nuclear envelope when they are formed in wild-type spermatocytes during the S3 stage, dany mutant spermatocytes are devoid of such characteristic nuclear envelope deformations at the corresponding stage. Thus, Dany appears to be required for normal chromatin association with the nuclear envelope. Since Dany is not enriched at the nuclear periphery, it obviously is very unlikely to function as a factor that directly establishes physical contact between chromatin and the nuclear envelope. But the extensive chromatin reorganization, which presumably occurs in early spermatocytes when thousands of previously repressed genes become active while precursor-specific genes are inactivated, might be abnormal in the absence of dany. Thereby, chromatin properties that normally cause localization to the nuclear periphery might also be affected (Trost, 2016).

Dany's closest relatives Dan and Danr, which act partially redundantly in somatic tissues, are crucial for the control of intranuclear position and silencing of the hunchback (hb) genomic locus in neuroblasts during embryogenesis. hb relocalization to the nuclear lamina, which is correlated with hb silencing and termination of Hb response competence, depends on timely Dan downregulation (Trost, 2016).

Changes in the association of genes with the nuclear periphery have previously been implicated in the control of the spermatocyte-specific gene expression program in Drosophila. In cell types other than spermatocytes, most spermatocyte-specific genes appear to be packaged into a repressive type of heterochromatin designated 'BLACK' heterochromatin, which is associated with Lam at the nuclear envelope. In case of two representative testis-specific gene clusters (at 60D1 and 22A1), loss of Lam was shown to result in their derepression in somatic larval and cultured cells, and the normal transcriptional activation was accompanied by cluster repositioning into the nuclear interior of late spermatocytes. Immuno-FISH analyses support the notion that a repressive chromatin type that is normally associated with the nuclear periphery is impaired in dany mutants. The derepression of the eye-specific inaC gene in dany mutant spermatocytes was accompanied by delocalization away from the nuclear periphery into the interior. Extensive future work will be required to clarify the molecular basis and functional significance of these initial observations (Trost, 2016).

Even if an extensive correlation between intranuclear position and transcriptional activity were established by systematic studies, it remains to be considered whether loss of dany might affect the organization of chromatin within the nucleus indirectly. dany mutations could alter the expression program of particular chromatin regulators or proteins of the nuclear periphery. Indeed, some important chromatin regulators and nuclear envelope proteins have been shown to undergo drastic changes in expression levels during normal spermatogenesis. Su(Hw), a multi-zinc finger DNA-binding protein that can function as a transcriptional insulator and modulator of chromatin association with the nuclear lamina, and the PRC2 complex components E(z) and Su(z) are strongly downregulated in early spermatocytes, whereas Lamin C (LamC) expression is induced. However, in these particular cases, no abnormal expression program was detected in dany mutant testis by immunolabeling (Trost, 2016).

Loss of dany also results in the derepression of genes that appear to be Pc targets. Such genes are primarily within 'BLUE' heterochromatin. Pc-mediated repression of spermatocyte-specific genes has been proposed to be counteracted in spermatocytes by the combined action of tMAC and tTAFs. Although not identical, the dany mutant phenotype shares similarities with the meiotic arrest phenotype caused by loss of tMAC and tTAF function. Future work will be required to clarify the functional interactions between Dany and these major activators of spermatocyte-specific genes (Trost, 2016).

Extensive spatial intranuclear chromatin reorganization might guide many differentiation processes in complex eukaryotes. For example, hundreds of genes were reported to move towards or from the nuclear envelope during differentiation of mouse embryonic stem cells, concomitant with reduced or increased expression, respectively. Similar observations were made during adipogenic cell differentiation. This identification and initial characterization of dany should help support future progress towards a molecular understanding of the mechanisms that coordinate change in spatial genome organization and cell type-specific expression programs (Trost, 2016).

Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. This study identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, these results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis (Wen, 2016).

The dynamic process of spermatogenesis shows little variation between invertebrate models such as Drosophila, and vertebrate models such as mice and rats. In each case, germ stem cells undergo mitotic division to proliferate and then continue, via meiosis, through various stages of elongation and individualization from spermatogonia to spermatid to finally to form mature sperm. Mature sperm are then stored in the seminal vesicles for fertilization. Errors in any of these stages can lead to male infertility. This study found that Drosophila Pif1A acts as a key regulator for sperm individualization. Loss of Pif1A leads to male sterility associated with irregular individualization complex and empty seminal vesicles without mature sperm. Pif1A is highly expressed in the testes of mated male adult flies and the Pif1A protein is expressed at a higher level in male than in female flies. Pif1A is homologous to mammalian coiled-coil domain-containing protein 157 (CCDC157), which is also enriched in the testes of humans and mice. Human CCDC157, with unknown function, was identified to be downregulated in men with idiopathic non-obstructive azoospermia (NOA). The function of Drosophila Pif1A was mapped during spermatogenesis, showing that Pif1A is essential for spermatide individualization and involved in the regulation of the lipid metabolism genes. These findings might be applicable for studying the function of CCDC157 in spermatogenesis and other aspects of human male fertility (Yuan, 2019).

Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. This study showed that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. Mob4 was shown to be required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. This study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization (Santos, 2023).

The Drosophila testis is an excellent system for studying the process from germ stem cells to motile sperm, including the proliferation of male germ cells, meiosis of primary spermatocytes, mitochondrial morphogenesis, and spermatid individualization. Previous work has demonstrated that ocnus (ocn) plays an essential role in male germ cell development. Among those genes and proteins whose expression levels were changed as a result of ocn knockdown, cytochrome c1-like (cyt-c1L) was downregulated significantly. This study shows that cyt-c1L is highly expressed in the testis of D. melanogaster. Knockdown or mutation of cyt-c1L in early germ cells of flies resulted in male sterility. Immunofluorescence staining showed that cyt-c1L knockdown testes had no defects in early spermatogenesis; however, in late stages, in contrast to many individualization complexes (ICs) composed of F-actin cones that appeared at different positions in control testes, no actin cones or ICs were observed in cyt-c1L knockdown testes. Furthermore, no mature sperm were found in the seminal vesicle of cyt-c1L knockdown testes whereas the control seminal vesicle was full of mature sperm with needle-like nuclei. cyt-c1L knockdown also caused abnormal mitochondrial morphogenesis during spermatid elongation. Excessive apoptotic signals accumulated in the base of cyt-c1L knockdown fly testes. These results suggest that cyt-c1L may play an important role in spermatogenesis by affecting the mitochondrial morphogenesis and individualization of sperm in D. melanogaster (Chen, 2023).

Spermatogenesis occurs inside a somatic cell enclosure. Sperm release, the most important final step and a target for contraceptives, has been extensively studied in fixed tissue preparations. This study provides a time-lapse description of the release process in Drosophila testis ex vivo. The spermatid tails exit the somatic enclosure and enter the testicular duct first, followed by the spermatid heads. Prior to this, individual spermatid heads attempt to invade the head cyst cell, and on each occasion they are repelled by a rapid and local F-actin polymerization response from the head cyst cell. The F-actin assembly involves N-WASp, D-WIP, and Arp2/3 complex and dissipates once the spermatid head retreats back into the fold. These findings revise the existing spermiation model in Drosophila and suggest that somatic cells can actively oppose mechanical cell invasion attempts using calibrated F-actin dynamics in situ (Dubey, 2016).

Senescence is accompanied by loss of reproductive functions. Reproductive ageing was studied in Drosophila melanogaster males and asked whether the expected decline in male reproductive success is due to diminished functionality of the male accessory gland (AG). The male AG produces the majority of seminal fluid proteins (SFPs) transferred to the female at mating. SFPs induce female postmating changes and are key to male reproductive success. Sge-dependent gene expression changes were measured for five representative SFP genes in males from four different age groups ranging from 1 to 6 weeks after eclosion. Simultaneously, male reproductive success was measured in postmating traits mediated by transfer of these five SFPs. A decreased was found in male SFP gene expression with advancing age and an accompanying decline in male postmating success. Hence, male reproductive senescence is associated with a decline in functionality of the male AG. While overall individual SFP genes decreased in expression, these results point towards the idea that the composition of an ejaculate might change with male age as the rate of change was variable for those five genes (Koppik, 2017).

Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. In Drosophila, most SFPs are produced in the accessory glands, which are composed of approximately 1,000 fertility-enhancing "main cells" and approximately 40 more functionally cryptic "secondary cells." Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might regulate more global SFP functions and proteome composition. Secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, this study reveals changes to the seminal proteome that surprisingly encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. These results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution (Hopkins, 2019).

The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. This study has investigated the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. Cv-c was shown to be required to maintain testis integrity during fly development. Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. It was also demonstrated that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development (Sotillos, 2022).

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have found network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. RNA-seq was performed in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. The male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. The results are housed online in a searchable database as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia (Vincent, 2019).

Despite holding a central role in fertilization, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis-signalling between the male ejaculate and the female during and after mating resulting in postmating prezygotic (PMPZ) isolation between populations. Crosses between Drosophila montana populations show PMPZ isolation in the form of reduced fertilization success in both noncompetitive and competitive contexts. This study tested whether male ejaculate proteins produced in the accessory glands or ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. More than 150 differentially abundant proteins were found between populations that may contribute to PMPZ isolation, including a number of proteases, peptidases and several orthologues of Drosophila melanogaster Sfps known to mediate fertilization success. Males from the population that elicit the stronger PMPZ isolation after mating with foreign females typically produced greater quantities of Sfps. The accessory glands and ejaculatory bulb show enrichment for different gene ontology (GO) terms and the ejaculatory bulb contributes more differentially abundant proteins. Proteins with a predicted secretory signal evolve faster than nonsecretory proteins. Finally, advantage was taken of quantitative proteomics data for three Drosophila species to determine shared and unique GO enrichments of Sfps between taxa and which potentially mediate PMPZ isolation. This study provides the first high-throughput quantitative proteomic evidence showing divergence of reproductive proteins between populations that exhibit PMPZ isolation (Garlovsky, 2020).

One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer (Rambur, 2020).

Worldwide, a large majority of cancers originates from epithelial tissues such as lung, breast and prostate1. Despite reinforced prevention, most of the tumours are detected at late stages, and patient care is centred on invasive adenocarcinomas, resistant forms of these carcinomas and metastatic carcinomas. As late stages of cancer progression have been under intense scrutiny in the last decades, the molecular mechanisms associated to such progression are largely described, showing for example the major role of receptor tyrosine kinase (RTK)-dependent signalling pathways in these mechanisms. Typically, for prostate adenocarcinoma, the second most common cancer in men, both PI3K/AKT/mTOR pathway and Ras/MAPK pathways are associated with tumour progression. In the prostate adenocarcinoma, Ras/MAPK and PI3K/AKT/mTOR pathways display activating genetic alterations in more than 40% of primary tumours and in virtually all metastatic prostate tumours, and phosphoproteomic studies confirmed a strong correlation in the activation of these two pathways. Furthermore, pre-clinical mouse models reproducing alteration of either one or the other pathway in the prostate epithelium display tumourigenesis that mimics histopathological features of the human adenocarcinoma. Moreover, advanced tumour progression is obtained when combining alterations in both pathways. These different data emphasise that in one hand, Ras/MAPK or PI3K/AKT/mTOR pathways can initiate prostate tumour development, and in the other hand that these pathways are implicated in late phases of tumour progression. However, they explain neither their respective or combined role in actual adenocarcinoma formation nor the molecular mechanisms that could couple these two pathways to promote this phenomenon in vivo (Rambur, 2020).

Adenocarcinoma formation occurs when pre-invasive epithelial cells acquire the ability to leave their epithelial compartment. This implicates that these cells are able to extrude from the normal epithelium and to cross the basement membrane which is the limit of the epithelial compartment. These phenomena can be described as basal extrusion and are resulting in early invasion, as opposed to late invasion associated to the metastatic process. Due to the difficulty to precisely visualise basal extrusion in animals, mechanistic associated to this phenomenon has been essentially described in cellular models or in developing tissues such as Drosophila imaginal disc of zebrafish embryo, even though the role of P120 catenin in basal extrusion has been shown in a mouse model of pancreatic neoplasia. The role of Ras/MAPK pathway in basal extrusion has only been described through the use of RasV12 as an oncogenic hit, and role of PI3K/AKT/mTOR pathway has never been assessed (Rambur, 2020).

To determine the role of Ras/MAPK and PI3K/AKT/mTOR pathways in basal extrusion and understand the underlying mechanisms that may coordinate their hyperactivation in prostate cancer, this study has developed a new model of in vivo early invasive adenocarcinoma in the Drosophila prostate-like accessory gland. Drosophila is a powerful genetic model where more than 70% of genes implicated in human diseases display orthologs and where Ras/MAPK and PI3K/AKT/mTOR signalling pathways are well conserved. Drosophila has already proven its pertinence as cancer model for brain, lung, and colon. The Drosophila accessory gland is a functional equivalent for the prostate, playing a role in fertility by secreting seminal fluid. Secretions come from a monolayer of epithelial cells that are well differentiated and quiescent at the adult age, and there is no evidence of stem cells in this tissue. Considering that a majority of prostate adenocarcinoma is thought to originate from luminal cells, epithelial cells from the accessory gland represent a valuable model to study the mechanisms of epithelial prostate tumourigenesis (Rambur, 2020).

This study describes this unique model of basal extrusion and tumour formation in the accessory gland that recapitulates most aspects of cancer development. Both Ras/MAPK and PI3K/Akt/TOR pathways are overactivated in the produced tumours, and these pathways cooperate to induce basal extrusion and subsequent tumour formation. Furthermore, the mechanism is described that allows the coactivation of these pathways, which relies on the sequential recruitment of a double autocrine feedback loop dependent on Epidermal (EGF/Spitz) and Insulin-like (IGF/Ilp6) Growth Factors and their respective receptors. Finally, using publicly available data of prostate cancer samples and migration assay in human pre-tumoural prostate epithelial cell line, the possible role of these findings in the actual human pathology is assessed (Rambur, 2020).

To faithfully reproduce what is thought to happen in the earliest stages of tumour formation in patients, a single genetic alteration was produced in few clones of randomly selected and mostly differentiated cells. Furthermore, accessory gland epithelium, shown to be adjacent to a basement membrane, is surrounded by a stromal-like sheet of muscle fibres, and oncogene-induced epithelial cells are able to cross both layers to form external tumours. This recapitulates the phenomenon of basal epithelial cell extrusion, which is thought to be central to cell invasion. Basal extrusion has been described in cell culture, in Drosophila imaginal discs, in zebrafish embryos and in mouse. However, implication of Ras/MAPK and PI3K/AKT/mTOR pathways has never been assessed in this phenomenon, despite the fact that these pathways are among the most deregulated in cancers, and especially in epithelial cancers such as prostate adenocarcinoma. This study shows in a new model of accessory gland tumourigenesis that both pathways are implicated in basal extrusion, indicating that this step demands a particular state of activation for the cell that undergoes this basal extrusion. Furthermore, this finding correlates with the fact that the two considered pathways are already frequently co-deregulated in primary tumours. From these experiments, where oncogene expression is restricted to few cells and intra-tumoural inhibition of the pathways decreases invasion, it is infered that the mechanisms of basal cell extrusion are cell autonomous, as previously shown in cell lines. Indeed, this study shows that this cell-autonomous mechanism relies on the production of two growth factors, and subsequent activation of two autocrine loops. Role of autocrine loops has been hypothetized in late tumourigenesis, as higher levels of growth factors have been found in tumoural tissues, and has been studied in cell models where inhibition of these loops decreases tumourigenic features such as migration or proliferation capacity as their activation have been linked to transformation of various epithelial cells. However, a role of autocrine loops has never been demonstrated for basal extrusion in vivo. If these loops seem implicated in tumour late progression, so could they be more important for early human tumour development. In fact, many strategies have been attempted to treat cancer patients especially by blocking EGF/EGFR autocrine loop. However, for advanced prostate cancer, these strategies have shown poor results, as well for monotherapies as for combined treatments with classical anti-prostate cancer agents. It could be logical if autocrine loops are less implicated in late stages of cancer but more in the capacity for tumour cells to leave the epithelial compartment. In later stages, higher rates of activating mutations in the Ras/MAPK and PI3K/AKT/mTOR pathways could suppress the need for RTK-driven activation. In contrast, in early tumourigenesis, as fewer genetic alterations are present, activation of signalling pathways must rely on different mechanisms. As is shown in the accessory gland, this recruitment could be efficiently done in tumour cells by autocrine production of growth factors, autocrine activation of their RTK and subsequent activation of the pathways necessary for the tumour development. In a human cohort of prostate cancer samples, it was found that EGF is more expressed in primary tumours than either in normal tissue or in metastases. This could correlate with an early requirement for such growth factor in the formation of adenocarcinoma. Contrary to the observations in Drosophila, no early overexpression of IGFs can be detected in human samples. However, in human, EGFR is able to recruit both Ras/MAPK and PI3K/AKT/mTOR pathway, and EGF overexpression could drive their activation and act in the same way as EGF/Spitz and IGF/Ilp6 in Drosophila (Rambur, 2020).

To study early phases of tumourigenesis remains difficult in vivo, especially for epithelial cells that can develop into benign tumours still in the epithelial compartment such as benign prostatic intraepithelial neoplasia, or into adenocarcinoma that are characterised by an expansion out of the epithelial compartment. The model developed in the Drosophila accessory gland represents a unique in vivo model to explore basal extrusion and early invasion. Two major pathways of cancer progression are implicated in this basal extrusion, and these two pathways are co-recruited by autocrine loops. Further investigation will be necessary to test whether other pathways implicated in late tumourigenesis are important in this phenomenon. Furthermore, it will also be important to determine which genes are activated or inhibited by these pathways and which mechanisms are recruited to promote the actual extrusion (Rambur, 2020).

One of the best examples of sexual dimorphism is the development and function of the gonads, ovaries and testes, which produce sex-specific gametes, oocytes, and spermatids, respectively. The development of these specialized germ cells requires sex-matched somatic support cells. The sexual identity of somatic gonadal cells is specified during development and must be actively maintained during adulthood. Previous work showed that the transcription factor Chinmo is required to ensure the male sexual identity of somatic support cells in the Drosophila melanogaster testis. Loss of chinmo from male somatic gonadal cells results in feminization: they transform from squamous to epithelial-like cells that resemble somatic cells in the female gonad but fail to properly ensheath the male germline, causing infertility. To identify potential target genes of Chinmo, somatic cells deficient for chinmo were purified from the adult Drosophila testis, and next-generation sequencing was performed to compare their transcriptome to that of control somatic cells. Bioinformatics revealed 304 and 1549 differentially upregulated and downregulated genes, respectively, upon loss of chinmo in early somatic cells. Using a combination of methods, several differentially expressed genes were validated. These data sets will be useful resources to the community (Grmai, 2021).

Sperm hyper-activation is a dramatic change in sperm behavior where mature sperm burst into a final sprint in the race to the egg. The mechanism of sperm hyper-activation in many metazoans, including humans, consists of a jolt of Ca2+ into the sperm flagellum via CatSper ion channels. Surprisingly, all nine CatSper genes have been independently lost in several animal lineages. In Drosophila, sperm hyper-activation is performed through the cooption of the polycystic kidney disease 2 (pkd2) Ca2+ channel. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the molecular evolution of the sperm hyper-activation machinery in two independent, nonhomologous calcium channels separated by > 500 million years of divergence. This study used a comprehensive phylogenomic approach to investigate the selective pressures on these sperm hyper-activation channels. First, it was found that the entire CatSper complex evolves rapidly under recurrent positive selection in primates. Second, it was found that pkd2 has parallel patterns of adaptive evolution in Drosophila. Third, this adaptive evolution of pkd2 is driven by its role in sperm hyper-activation. These patterns of selection suggest that the evolution of the sperm hyper-activation machinery is driven by sexual conflict with antagonistic ligands that modulate channel activity (Cooper, 2017).

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. This study performed a large-scale CRISPR/Cas9 mutagenesis of 'conserved, essential' and 'young, RNAi-lethal' genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, widespread signatures of positive selection were detected on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function (Kondo, 2017).

In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. This study reports a gene named sheepish (shps) of D. melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female post-mating responses after copulation with shps males appeared normal at least with respect to conformational changes of uterus, mating plug formation and female remating rates. The shps gene encodes a protein with homology to amine oxidases, including as observed in mammals, with a transmembrane region at the C-terminal end. The shps mutation was characterized by a nonsense replacement in the third exon of CG13611 and shps was rescued by transformants of the wild-type copy of CG13611 Thus, shps may define a new class of gene responsible for sperm storage (Tomaru, 2017).

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. This study investigated the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. This study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, it is proposed that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline (Lu, 2018).

To study the regulatory mechanism of the Aryl hydrocarbon receptor (AHR), target genes of transcription are necessary for understanding the normal developmental and pathological processes. This study examined the effects of human AHR ligands on male fecundity. To induce ectopic human AhR gene expression, Drosophila melanogaster was used that was transformed with human AhR under the control of a yeast UAS promoter element capable of activation in the two-component UAS-GAL4 system. Exogenous AHR ligands decrease the number of Drosophila gonadal Traffic jam-positive cells. Both an increase and decrease was observed of AHR target gene expression, including in genes that control homeostasis and testis development. This suggests that gonadal AHR activation may affect the expression of gene networks that control sperm production and could be critical for fertility not just in Drosophila but also in humans. Finally, the activation of the expression for some AHR target genes depends on the expression of testis-specific chaperone CG5017 in gonadal cells. Since CG5017 belongs to the nucleosome assembly protein (NAP) family and may participate in epigenetic regulation, it is proposed that this nucleotropic chaperone is essential to provide the human AHR with access to only the defined set of its target genes during spermatogenesis (Akishina, 2018).

Spermatogenesis in Drosophila is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. This study addressed how distinct subsets of target genes are selected. The highly similar proteins tPlus3a and tPlus3b (CG31702 and CG31703) that contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes, were. characterized. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants were determined using next-generation sequencing (RNA-seq), and they were compared to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5, which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. It is hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes (Hundertmark, 2019).

Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. This study identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. Two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. Two RNA-binding proteins were identified that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. It is proposed that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation (Fingerhut, 2019).

The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, single-cell RNA-sequencing was performed of adult Drosophila testis. New genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation (Witt, 2019).

Sperm competition favors large, costly ejaculates, and theory predicts the evolution of allocation strategies that enable males to plastically tailor ejaculate expenditure to sperm competition threat. While greater sperm transfer in response to a perceived increase in the risk of sperm competition is well-supported, there is only a poor understanding of whether males (i) respond to changes in perceived intensity of sperm competition, (ii) use the same allocation rules for sperm and seminal fluid, and (iii) experience changes in current and future reproductive performance as a result of ejaculate compositional changes. Combining quantitative proteomics with fluorescent sperm labeling, this study shows that Drosophila melanogaster males exercise independent control over the transfer of sperm and seminal fluid proteins (SFPs) under different levels of male-male competition. While sperm transfer peaks at low competition, consistent with some theoretical predictions based on sperm competition intensity, the abundance of transferred SFPs generally increases at high competition levels. However, it was found that clusters of SFPs vary in the directionality and sensitivity of their response to competition, promoting compositional change in seminal fluid. By tracking the degree of decline in male mating probability and offspring production across successive matings, evidence is provided that ejaculate compositional change represents an adaptive response to current sperm competition, but one that comes at a cost to future mating performance. This work reveals a previously unknown divergence in ejaculate component allocation rules, exposes downstream costs of elevated ejaculate investment, and ultimately suggests a central role for ejaculate compositional plasticity in sexual selection (Hopkins, 2019).

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. This study used the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. Carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. This work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized (Hudry, 2019).

Males and females differ in their physiology and disease susceptibility yet the sex of cells and animals has often been neglected in research, or a single sex (male) is preferentially used. This might have prevented identification of sex differences that could inform clinical studies and therapies. Pressure to consider both sexes in basic and clinical research is revealing that sex differences are extensive, yet relatively underexplored (Hudry, 2019).

Sex chromosome sensing in Drosophila melanogaster activates a splicing cascade that results in expression of the RNA-binding protein Tra^F only in females, leading to sex-specific splicing of the transcription factors Doublesex (Dsx) and Fruitless (Fru) in a subset of cells, which sculpt sexually dimorphic anatomical features, reproductive systems, and behavior. Although superficially distinct from mammalian mechanisms involving gonadal release of sex hormones, Drosophila and mammalian sex differentiation shares common effectors such as the Dmrt/Dsx family of transcription factors. Furthermore, mouse models have revealed a cell-intrinsic contribution of sex chromosome complements to sex differences in body size and adiposity in mammals, and studies in flies have hinted at cell-extrinsic contributions to sex-biased phenotypes. Thus, sex differentiation in both insects and mammals appears to be a complex process integrating intrinsic and extrinsic inputs (Hudry, 2019).

Like its mammalian counterpart, the adult Drosophila digestive tract is a plastic and functionally regionalized organ, harboring microbiota and cell types akin to those found in humans, including self-renewing epithelial progenitors, digestive and absorptive enterocytes (ECs), and hormone-secreting enteroendocrine cells.Recently work has revealed sex differences in intestinal stem cell proliferation, which are adult-reversible and intrinsic to the stem cells (Hudry, 2016). During the course of these experiments, intestinal sex differences were observed in metabolic gene expression (Hudry, 2016), suggesting that sex-biased intestinal metabolism might contribute to sex differences in whole-body physiology (Hudry, 2019).

The intestine communicates with other organs, and peptide hormones are well established mediators. However, intermediate products of intracellular, housekeeping metabolic pathways are detected in the circulation, and recent work is revealing that both healthy tissues and tumors can use (and sometimes require) such exogenous, circulating metabolites. Consequently, there is considerable interest in exploring the instructive potential of metabolites in the context of inter-organ signaling (Hudry, 2019).

This study has uncover bi-directional communication between the male gonad and an adjacent intestinal region. This communication affects both gut and testes function and is mediated by cytokine signaling and the metabolite citrate (Hudry, 2019).

Regional differences in gene expression are observed along animal gastrointestinal tracts, suggestive of functional specializations. This study now provides evidence for region- and cell-type-specific carbohydrate metabolism. Intestinal carbohydrate metabolism also differs between the sexes, illustrating how sex differences can be confined to specific organ portions; even when digestive enzymes are more broadly expressed along the midgut, their male upregulation is posterior midgut (R4)-specific. It is suggested that specific gut portions might be physiologically 'sexualized' to subserve reproductive needs -- in this case spermatogenesis. The posterior midgut might be more broadly sexually dimorphic than other intestinal regions; oxidative stress response proteins are male biased and Yp1 is female biased in this same region (Hudry, 2016). In female flies, posterior midgut ECs adjust their lipid metabolism after mating to maximize reproductive output. It will be of interest to explore whether this requires their female identity; if it does, is female identity the 'ground state' in the absence of a male gonad, or does it result from an ovary signal? Comparative studies could also explore contributions of intestinal sex differences to reproductive success in animals other than Drosophila and whether the evolution of a placenta (an organ purpose-built for reproduction) replaced or reinforced such intestinal contributions in female mammals (Hudry, 2019).

The male gonad controls sex differences in intestinal carbohydrate metabolism through male-biased cytokine signaling activity. Drosophila Upd proteins belong to the type I family of cytokines, like mammalian interleukins and leptin. In both humans and rodents, leptin expression is sexually dimorphic. Males and females also differ in their interleukin repertoire, which contributes to sex differences in immunity and autoimmune disease. A possible contribution of cytokines such as leptin to sex differences in organ physiology deserves further investigation, particularly in light of leptin's known reproductive and gastrointestinal roles (Hudry, 2019).

The gonadal regulation of intestinal sugar metabolism contrasts with the intrinsic, sex-chromosome-dependent control of sex differences in gut stem cell proliferation (Hudry, 2016). This illustrates the complexity of an organ's 'sexual identity;' two lineage-related cells within an epithelium (stem cells and their EC progeny) acquire sex-specific functions (proliferation and carbohydrate metabolism) through two distinct mechanisms. Sexual identity is reversible in both cases and needs to be actively maintained in adults, raising the question of whether adult plasticity in sexual identity might be adaptive. Environmental factors could modulate the expression or penetrance of sex determinants -- possibly tissue specifically. There is some evidence in support of this idea: male flies that lack Fru^M are defective in courtship but learn to court when housed in groups with wild-type flies in a Dsx^M-dependent manner. Early life exposure to nutrient scarcity also affects neuronal wiring selectively of male C. elegans. In light of these findings, it will be of interest to explore how plastic sex differences in physiology are and why (Hudry, 2019).

Gut-gonad communication is bi-directional; the male gonad communicates with a specific gut portion, which responds by secreting citrate. Gut-derived citrate in turn promotes food intake and maturation of male gametes. How might it do so? Import of exogenous citrate might help sustain the high TCA cycle requirements of developing sperm. Sertoli cells are highly glycolytic and have been proposed to act as a paracrine source of lactate for developing gametes. It is therefore conceivable that citrate acts as another exogenous carbon source. Consistent with this idea, the mitochondrial citrate carrier is present and active in human sperm, and boar sperm can metabolize exogenous citrate through the Krebs cycle in vitro. Alternatively, import of gut-derived citrate might sustain membrane formation through its conversion to acetyl-CoA by ATCPL, then used for fatty acid synthesis; both spermatid elongation and individualization require extensive membrane biosynthesis and remodeling. Citrate could also support epigenetic changes relevant to male gamete maturation through its conversion to acetyl-CoA, used as a donor for histone acetyl transferase-mediated histone acetylatione (Hudry, 2019).

The effects of gut-derived citrate on sperm production can be uncoupled from its orexigenic actions. Preventing citrate import into neurons reduces food intake, suggesting that its promotion of feeding might result from its actions in the nervous system. Given that preventing gut-derived citrate efflux does not affect circulating citrate, it is tempting to speculate that local gut and/or testis-innervating neurons might harbor the citrate sensors. This effect of citrate on food intake is male specific: reducing gut-derived citrate efflux does not reduce feeding in females. This ongoing work is revealing that, in females, gonad to gut communication also promotes feeding, but via a different mechanism and possibly as a result of different dynamics and/or metabolic requirements of male and female gamete production (Hudry, 2019).

More generally, this study provides evidence that citrate functions in communication between organs. In mammals, plasma levels of citrate are among the highest among TCA cycle intermediates. Organ-specific differences in citrate production and consumption have been reported, but little is known about its roles and regulation by diet, age, or sex. Bone, an organ that controls male fertility through an endocrine hormone, produces unusually high amounts of citrate. In the context of male gametes, the prostate should also be considered as a potentially relevant citrate source; it secretes large amounts of citrate into the seminal fluid that developing sperm will come into contact with. The roles of prostate citrate have been investigated in the context of the metabolic rewiring of prostate tumors. Less is known about its roles in the context of sperm production, partly because surgical interventions such as prostatectomy impair other aspects of testis physiology. Contributions of exogenous citrate to sperm-mediated transgenerational effects also deserve further investigation in light of citrate's epigenetic effects. It will also be of interest to characterize the transporters for citrate import into the germline to control spermatogenesis and/or into neurons to control food intake; CG7309 and Indy-2 genes code for putative citrate transporters and have testis-specific expression. In mammals, the Indy homolog NaCT is specifically expressed in testis, liver, and brain, and NaCT knockout mice are protected from diet- and age-induced adiposity and insulin resistance (Hudry, 2019).

The physical proximity between the male gonad and the gut portion to which it signals raises the possibility that the relative positioning of internal organs is physiologically significant. Although this particular association is not conserved in adult humans, testis development is a complex process from a three-dimensional perspective, which in all placental mammals involves descent of testes from a position near the kidneys, perhaps providing opportunities for inter-organ communication. More generally, a spectrum of conditions (so-called heterotaxy syndromes) resulting from the abnormal arrangement of internal organs including the gastrointestinal tract can lead to serious disease manifestations. Subtler, likely undiagnosed defects in intestinal positioning could result in milder gastrointestinal symptoms and/or contribute to differences in whole-body physiology across individuals (Hudry, 2019).

Metabolic profiles vary across all levels of biological diversity, from cells to taxa. Two-photon fluorescence lifetime imaging microscopy (FLIM) facilitates metabolic characterisation of biological specimens by assaying the intrinsic autofluorescence of the ubiquitous coenzymes NAD(P)H and FAD. The potential of this method for characterising the diversity of organismal metabolism remains largely untapped. Using FLIM in Drosophila melanogaster, this study shows tissue-specificity in fluorescence lifetime that reflects variation in redox patterns. In particular, sperm cells exhibited elevated glycolysis relative to other tissues. Sperm metabolism was shown to be phenotypically plastic: compared to male-stored sperm, sperm stored in the female's storage organ showed a substantial reduction in the protein-bound FAD lifetime fraction but no change in the NAD(P)H profile. This study represents the first ex vivo investigation of sperm metabolism using FLIM (Wetzker, 2019)

Sperm viability is a major male fitness component, with higher sperm viability associated with enhanced sperm competitiveness. While many studies have focussed on sperm viability from the male fitness standpoint, its impact on female fitness is less clear. This study used a panel of 32 isogenic Drosophila simulans lines to test for genetic variation in sperm viability (percentage of viable cells). Then, whether sperm viability affected female fitness was tested by mating females to males from low or high sperm viability genotypes. Significant variation was found in sperm viability among genotypes, and consistent with this, sperm viability was highly repeatable within genotypes. Additionally, females mated to high sperm viability males laid more eggs in the first seven hours after mating, and produced more offspring in total. However, the early increase in oviposition did not result in more offspring in the 8 hours following mating, suggesting that mating with high sperm-viability genotypes leads to egg wastage for females shortly after copulation. Although mating with high sperm-viability males resulted in higher female fitness in the long term, high quality ejaculates would result in a short-term female fitness penalty, or at least lower realised fitness, potentially generating sexual conflict over optimal sperm viability (Tourmente, 2019).

This study tested how exposure to different average temperatures that either fluctuated or remained constant impacts different male reproductive performance traits and to assess adaptive potential to future heat stress. Advantage was taken of a set of Drosophila melanogaster isogenic lines of different genotypes, exposing them to four different thermal conditions. Subsequent male reproductive performance was measured for mating success, fertility, number of offspring produced, and offspring sex ratio, and the influence of thermal stress on estimated heritability and evolvability of these reproductive traits was calculated. Both costs and benefits to different thermal conditions on reproductive performance were found, with some responses varying between genotypes. Mating success improved under fluctuating benign temperature conditions and declined as temperature stress increased regardless of genotype. Fertility and productivity were severely reduced at fluctuating mean high temperature for all genotypes, but some genotypes were unaffected at constant high mean temperature. These more thermally robust genotypes showed a slight increase in productivity under the fluctuating benign condition compared to constant high temperature, despite both thermal conditions sharing the same temperature six hours daily. Increasing thermal stress resulted in higher heritability and evolvability. Overall, the effects of temperature on reproductive performance depended on the trait and genotype; performance of some traits slightly increased when high temperatures were experienced for short periods but decreased substantially even when experiencing a benign temperature for a portion of each day. While thermal stress increased genetic variation that could provide adaptive potential against climate warming, this is unlikely to compensate for the overall severe negative effect on reproductive performance as mean temperature and variance increase (Rodrigues, 2021).

Dietary fatty acids can accumulate in sperm and affect their function in vertebrates. There is currently no clear prediction as to how dietary fatty acids affect the sperm of D. melanogaster. This study manipulated the amount and identity of dietary polyunsaturated fatty acids (PUFA) in the food of D. melanogaster males (a treatment known to affect membrane fluidity) and measured changes in sperm parameters. The following foundings were made: (a) males reared on food containing PUFA-rich, plant-derived lipids showed a slower increase in sperm volume over male age compared to males reared on yeast-derived lipid food which is richer in saturated fatty acids. (b) The resistance of sperm membrane integrity to osmotic stress was not altered by dietary lipid treatment, but (c) food containing yeast-derived lipids induced a 46% higher in situ rate of production of reactive oxygen species in sperm cells. These findings show that dietary lipids have similar effects on sperm parameters in Drosophila as in vertebrates, affect some, but not all, sperm parameters and modulate male reproductive ageing. In concert with recent findings of sex-specific seasonal variation of diet choice in the wild, these results suggest a substantial dietary impact on the dynamics of male reproduction in the wild (Guo, 2020).

The Y chromosome of Drosophila melanogaster is pivotal for male fertility. Yet, only 16 protein-coding genes reside on this chromosome. The Y chromosome is comprised primarily of heterochromatic sequences, including DNA repeats and satellite DNA, and most of the Y chromosome is still missing from the genome sequence. Furthermore, the functions of the majority of genes on the Y chromosome remain elusive. Through multiple genetic strategies, six distinct segments on the Y chromosome have been identified as "male fertility factors," and candidate gene sequences corresponding to each of these loci have been ascribed. In one case, kl-3, a specific protein coding sequence for a fertility factor has been confirmed molecularly. This study employed CRISPR/Cas9 to generate mutations, and RNAi, to interrogate the requirements of protein coding sequences on the Y chromosome for male fertility. CRISPR/Cas9-mediated editing of kl-2 and kl-5 causes male sterility, supporting the model that these gene sequences correspond to the cognate fertility factors. Another gene, CCY, also functions in male fertility and may be the ks-2 fertility factor. This study demonstrates that editing of kl-2, kl-3 and kl-5, and RNAi knockdown of CCY, disrupts nuclear elongation, and leads to defects in sperm individualization, including impairments in the individualization complex (IC) and synchronization. However, CRISPR/Cas9 mediated knockout of some genes on the Y chromosome, such as FDY, Ppr-Y and Pp1-Y2 do cause sterility, indicating that not all Y chromosome genes are essential for male fertility (Zhang, 2020).

Animal germ cells communicate directly with each other during gametogenesis through intercellular bridges, often called ring canals (RCs), that form as a consequence of incomplete cytokinesis during cell division. Developing germ cells in Drosophila have an additional specialized organelle connecting the cells called the fusome. Ring canals and the fusome are required for fertility in Drosophila females, but little is known about their roles during spermatogenesis. With live imaging, this study directly observed the intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis, from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids, demonstrating that RCs are stable and open to intercellular traffic throughout spermatogenesis. Disruption of the fusome, a large cytoplasmic structure that extends through RCs and is important during oogenesis, had no effect on spermatogenesis or male fertility under normal conditions. Our results reveal that male germline RCs allow the sharing of cytoplasmic information that might play a role in quality control surveillance during sperm development (Kaufman, 2020).

Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4(th)) that re-acquired autosomal status. This study used single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4(th) chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis that were identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5 (Mahadevaraju, 2021).

The spectrin cytoskeleton has been shown to be critical in diverse processes such as axon development and degeneration, myoblast fusion, and spermatogenesis. Spectrin can be modulated in a tissue specific manner through junctional protein complexes, however, it has not been shown that long noncoding RNAs (lncRNAs) interact with and modulate spectrin. This study provides evidence of a lncRNA CR45362 that interacts with α-Spectrin, is required for spermatid nuclear bundling during Drosophila spermatogenesis. It was observed that CR45362 showed high expression in the cyst cells at the basal testis, and CRISPR-mediated knockout of CR45362 led to sterile male, unbundled spermatid nuclei, and disrupted actin cones. Through chromatin isolation by RNA precipitation-mass spectrometry (ChIRP-MS), actin-spectrin cytoskeletal components were observed tophysically interact with the lncRNA CR45362. Genetic screening on identified cytoskeletal factors revealed that cyst cell-specific knockdown of α-Spectrin phenocopied CR45362 mutants and resulted in spermatid nuclear bundle defects. Consistently, CR45362 knockout disrupted the co-localization of α-Spectrin and spermatid nuclear bundles in the head cyst cells at the basal testis. Thus, this study uncovered a novel lncRNA CR45362 that interacts with α-Spectrin to stabilize spermatid nuclear bundles during spermatid maturation (Bouska, 2021).

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. This study comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, new piRNA clusters were identified, and complex satellites were identified as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. Two piRNA clusters were identified that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene (Chen, 2021).

Evolutionarily young genes are usually preferentially expressed in the testis across species. While it is known that older genes are generally more broadly expressed than younger genes, the properties that shaped this pattern are unknown. Older genes may gain expression across other tissues uniformly, or faster in certain tissues than others. Using Drosophila gene expression data, this study confirmed previous findings that younger genes are disproportionately testis-biased and older genes are disproportionately ovary-biased. The relationship between gene age and expression is stronger in the ovary than any other tissue, and weakest in testis. This study performed ATAC-seq on Drosophila testis and found that while genes of all ages are more likely to have open promoter chromatin in testis than in ovary, promoter chromatin alone does not explain the ovary-bias of older genes. Instead, it was found that upstream transcription factor (TF) expression is highly predictive of gene expression in ovary, but not in testis. In ovary, TF expression is more predictive of gene expression than open promoter chromatin, whereas testis gene expression is similarly influenced by both TF expression and open promoter chromatin. It is proposed that the testis is uniquely able to expresses younger genes controlled by relatively few TFs, while older genes with more TF partners are broadly expressed with peak expression most likely in ovary. The testis allows widespread baseline expression that is relatively unresponsive to regulatory changes, whereas the ovary transcriptome is more responsive to trans-regulation and has a higher ceiling for gene expression (Witt, 2021).

Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. This study shows that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. This analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. It is proposed that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization (Herbette, 2021).

The Drosophila repleta group comprises more than one hundred species that inhabit several environments in the Neotropics and use different hosts as rearing and feeding resources. Rather homogeneous in their external morphology, they are generally distinguished by the male genitalia, seemingly their fastest evolving morphological trait, constituting an excellent model to study patterns of genital evolution in the context of a continental adaptive radiation. Although much is known about the evolution of animal genitalia at population level, surveys on macroevolutionary scale of this phenomenon are scarce. This study used a suite of phylogenetic comparative methods to elucidate the macroevolutionary patterns of genital evolution through deep time and large continental scales. The results indicate that male genital size and some aspects of shape have been evolving by speciational evolution, probably due to the microevolutionary processes involved in species mate recognition. In contrast, several features of the aedeagus (loosely compared to the penis) shape seemed to have evolved in a gradual fashion, with heterogeneous evolutionary phenotypic rates among clades. In general, the tempo of the evolution of aedeagus morphology was constant from the origin of the group until the Pliocene, when it accelerated in some clades that diversified mainly in this period. The incidence of novel ecological conditions in the tempo of aedeagus evolution and the relationship between species mate recognition and speciation in the Drosophila repleta group are discussed (Stefanini, 2021).

Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. This story focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. This study examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, this study induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, this study concluded that the Nep4 gene is essential for sperm function following sperm transfer to females (Ohsako, 2021).

Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. This study shows that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, this study shows that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. It is suggested that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns (Pu, 2021).

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. This study provides an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and this study provides the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. Nine of the 16 Y-linked proteins were identified, including known testis-specific male fertility factors. Almost one-half of known Drosophila ribosomal proteins were identified in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, the expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. A significant fraction of 'sperm-associated Sfps' are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, these results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution (Garlovsky, 2022).

Wolbachia is an obligate intracellular bacterium that can alter reproduction of its arthropod hosts, often through a mechanism called cytoplasmic incompatibility (CI). In CI, uninfected females fertilized by infected males yield few offspring, but if both are similarly infected, normal embryo viability results (called "rescue"). CI factors (Cifs) responsible for CI are pairs of proteins encoded by linked genes. The downstream gene in each pair encodes either a deubiquitylase (CidB) or a nuclease (CinB). The upstream gene products, CidA and CinA, bind their cognate enzymes with high specificity. Expression of CidB or CinB in yeast inhibits growth, but growth is rescued by expression of the cognate CifA protein. By contrast, transgenic Drosophila male germ line expression of both cifA and cifB was reported to be necessary to induce CI-like embryonic arrest; cifA expression alone in females is sufficient for rescue. This pattern, seen with genes from several Wolbachia strains, has been called the "2-by-1" model. This study shows that male germ line expression of the cinB gene alone, from a distinct clade of cif genes from wNo Wolbachia, is sufficient to induce nearly complete loss of embryo viability. This male sterility is fully rescued by cognate cinA(w)(No) expression in the female germ line. The proteins behave similarly in yeast. CinB(w)(No) toxicity depends on its nuclease active site. These results demonstrate that highly divergent CinB nucleases can induce CI, that rescue by cognate CifA factors is a general feature of Wolbachia CI systems, and that CifA is not strictly required in males for CI induction (Sun, 2022).

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. This study used cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild-type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers; however, Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, these results strongly support the Host modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue (Kaur, 2022).

Female mating rates vary widely, even among closely related species, but the reasons for this variation are not fully understood. Across Drosophila species, female mating frequencies are positively associated with sperm length. This association may be due in part to sperm limitation, with longer-spermed species transferring fewer sperm, or to cryptic female choice. However, a previously overlooked factor is sperm metabolic rate, which may correlate with sperm length. If faster-metabolizing sperm accumulate age-related cellular damage more quickly, then females should remate sooner to obtain fresh sperm. Alternatively, frequent female mating may select for increased sperm competitiveness via increased metabolism. This study measured sperm metabolism across 13 Drosophila species and compared these measures to published data on female mating rate and on sperm length. Using fluorescent lifetime imaging microscopy, this study quantified NAD(P)H metabolism ex vivo, in intact organs. Phylogenetically controlled regression reveals that sperm metabolic rate is positively associated with sperm length and with female mating frequency. Path analysis shows sperm length driving sperm metabolism and sperm metabolism either driving or being driven by female mating rate. While the causal directionality of these relationships remains to be fully resolved, and the effect of sperm metabolism on sperm aging and/or sperm competitiveness remains to be established, these results demonstrate the importance of sperm metabolism in sexual selection (Turnell, 2022).

Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The peripheral sensory information required to regulate copulation duration is unclear. This study employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. T these MSNs are cholinergic and co-express both fru and dsx. The sensory information received by the peripheral sensory neurons from the front legs (GRNs) and mechanosensory neurons (MSNs) at the male genitalia contribute to the regulation of copulation duration. Moreover, the results show that their function is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete (Jois, 2022).

Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. Focus was placed on one such gene, Rack 1, and it was found to play an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful (Ibaraki, 2021).

In species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, semiquantitative proteomics and sex-specific isotopic labeling were used in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. These data reveal a molecular "hand-off" from males to females, which is postulated to be an important component of sperm-female interactions (McCullough, 2022).

The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, this study applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO(2)) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. 182 phosphopeptides were identified, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis (Mao, 2022).

The impact of rising global temperatures on survival and reproduction is putting many species at risk of extinction. In particular, it has recently been shown that thermal effects on reproduction, especially limits to male fertility, can underpin species distributions in insects. However, the physiological factors influencing fertility at high temperatures are poorly understood. Key factors that affect somatic thermal tolerance such as hardening, the ability to phenotypically increase thermal tolerance after a mild heat shock, and the differential impact of temperature on different life stages are largely unexplored for thermal fertility tolerance. This study examine the impact of high temperatures on male fertility in the cosmopolitan fruit fly Drosophila virilis. It was first determined whether temperature stress at either the pupal or adult life history stage impacts fertility. Then the capacity for heat-hardening to mitigate heat-induced sterility was tested. Thermal stress was found to reduce fertility in different ways in pupae and adults. Pupal heat stress delays sexual maturity, whereas males heated as adults can reproduce initially following heat stress, but become sterile within seven days. Evidence was also found that while heat-hardening in D. virilis can improve high temperature survival, there is no significant protective impact of this same hardening treatment on fertility. These results suggest that males may be unable to prevent the costs of high temperature stress on fertility through heat-hardening, which limits a species' ability to quickly and effectively reduce fertility loss in the face of short-term high temperature events (Walsh 2021).

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