The Interactive Fly
Genes involved in tissue and organ development
INDEX
In the Drosophila oocyte, meiosis is arrested in the first division of metaphase, when a tapered spindle aligned parallel to the egg surface forms. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).
The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).
The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).
Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein, Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the absence of Ncd (Endow, 1998).
A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabillize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).
In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1; see Drosophila Tor pathway) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. This study defined opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. As is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Missing oocyte (Mio) and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. These studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. The data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms (Wei, 2014b).
In yeast, the inhibition of the nutrient-sensitive target of rapamycin complex 1 (TORC1) in response to amino acid limitation is essential for cells to transit from the mitotic cycle to the meiotic cycle. In response to amino acid starvation, the Iml1 complex, comprising the Iml1, Nitrogen permease regulator-like 2 (Npr2), and Nitrogen permease regulator-like 3 (Npr3) proteins in yeast and the respective orthologs DEPDC5, Nprl2, and Nprl3 in mammals, inhibits TORC1 activity. The Iml1 complex, which has been renamed the 'GTPase-activating proteins toward Rags 1' (GATOR1) complex in higher eukaryotes, functions as a GTPase-activating protein complex that inactivates RagsA/B or Gtr1 in mammals and yeast, respectively, thus preventing the activation of TORC1. In the yeast Saccharomyces cerevisiae, mutations in the Iml1 complex components Npr2 and Npr3 result in a failure to down-regulate TORC1 activity in response to amino acid starvation and block meiosis and sporulation. As is observed in yeast, in Drosophila, Nprl2 and Nprl3 mediate a critical response to amino acid starvation (Wei, 2014a). However, their roles in meiosis and gametogenesis remain unexplored (Wei, 2014b).
Recent reports indicate that the Iml1, Npr2, and Npr3 proteins are components of a large multiprotein complex originally named the 'Seh1-associated' (SEA) complex in budding yeast and the 'GATOR' complex in higher eukaryotes. The SEA/GATOR complex contains eight highly conserved proteins. The three proteins described above, Iml1/DEPDC5, Npr2/Nprl2, and Npr3/Nprl3, form the Iml1/GATOR1 complex and inhibit TORC1. The five remaining proteins in the complex, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59, which have been designated the 'GATOR2' complex in multicellular organisms, oppose the activity of Iml1/GATOR1 and thus promote TORC1 activity (Wei, 2014b).
Little is known about the physiological and/or developmental requirements for the GATOR2 complex in multicellular organisms. However, in Drosophila the GATOR2 components Mio and Seh1 interact physically and genetically and exhibit strikingly similar ovarian phenotypes, with null mutations in both genes resulting in female sterility (Senger, 2011; Wei, 2014a). In Drosophila females, oocyte development takes place within the context of an interconnected germline syncytium, also referred to as an 'ovarian cyst'. Ovarian cyst formation begins at the tip of the germarium when a cystoblast, the daughter of a germline stem cell, undergoes four synchronous divisions with incomplete cytokinesis to produce 16 interconnected cells. Actin-stabilized cleavage furrows, called 'ring canals', connect cells within the cyst. Each 16-cell cyst develops with a single oocyte and 15 polyploid nurse cells which ultimately are encapsulated by a somatically derived layer of follicle cells to produce an egg chamber. Each ovary is comprised of ~15 ovarioles that consist of a single germarium followed by a string of egg chambers in successively older stages of development. In mio- and seh1-mutant egg chambers, the oocyte enters the meiotic cycle, but as oogenesis proceeds, the oocyte fate and the meiotic cycle are not maintained stably (Senger, 2011; Wei, 2014a). Ultimately, a large fraction of mio and seh1 oocytes enter the endocycle and develop as polyploid nurse cells. A mechanistic understanding of how mio and seh1 influence meiotic progression and oocyte fate has remained elusive (Wei, 2014b).
This study demonstrates that the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. Depleting iml1 in the female germ line delays the mitotic/meiotic transition, so that ovarian cysts undergo an extra mitotic division. Conversely, mutations in Tor result in premature meiotic entry before the completion of the four mitotic divisions. Moreover, it was demonstrated that in the female germ line, the GATOR2 components Mio and Seh1 are required to oppose the TORC1 inhibitory activity of the Iml1/GATOR1 complex to prevent the constitutive down-regulation of TORC1 activity in later stages of oogenesis. These studies represent the first examination of the regulatory relationship between Iml1/GATOR1 and GATOR2 components within the context of a multicellular animal. Finally, these data reveal a surprising tissue-specific requirement for the GATOR2 complex in multicellular organisms and suggest a conserved role for the SEA/GATOR complex in the regulation of TORC1 activity during gametogenesis (Wei, 2014b).
Previous work demonstrated that in Drosophila the Iml1/GATOR1 complex mediates an adaptive response to amino acid starvation. This study tested the hypothesis that the Iml1/GATOR1 complex also has retained a role in the regulation of the early events of gametogenesis. Consistent with this model, this study found that in germline knockdowns of iml1, ovarian cysts delay meiotic entry and undergo a fifth mitotic division. This meiotic delay can be suppressed with the TORC1 inhibitor rapamycin. Thus, during Drosophila oogenesis the Iml1/GATOR1 complex promotes the transition from the mitotic cycle to the meiotic cycle through the down-regulation of the metabolic regulator TORC1. Increasing TORC1 activity by disabling its inhibitor delays meiotic progression, whereas germline clones of a Tor-null allele enter meiosis prematurely. Taken together, these data indicate that the level of TORC1 activity contributes to the timing of the mitotic/meiotic switch in Drosophila females and suggest that low TORC1 activity may be a conserved feature of early meiosis in many eukaryotes (Wei, 2014b).
However, in Drosophila, meiotic entry is not contingent on amino acid limitation at the organismal level. Indeed, the energy-intensive process of Drosophila oogenesis is curtailed dramatically when females do not have access to a protein source. Thus, to promote meiotic entry, Drosophila females must activate the Iml1/GATOR1 complex in a tissue-specific manner, using a mechanism that is independent of the overall nutrient status of the animal. At least two models can explain how Drosophila females might activate the Iml1/GATOR1 complex specifically in the germ line. In the first model, ovarian cysts locally experience low levels of amino acids during the mitotic cyst divisions and/or at the point of meiotic entry. These low levels of amino acids could reflect a non–cell-autonomous effect: The somatically derived escort cells that surround dividing ovarian cysts may function to create a low amino acid environment that triggers the activation of the Iml1/GATOR1 complex within developing ovarian cysts. Alternatively, the effect may be cell autonomous: The germ cells within dividing ovarian cysts may have a reduced ability to sense and/or import amino acids. In a second model, a developmental signaling pathway that is completely independent of local or whole-animal amino acid status directly activates the Iml1/GATOR1 complex. The identification of the upstream requirements for Iml1/GATOR1 activation in the female germ line will help distinguish between these two models (Wei, 2014b).
Although low TORC1 activity is required during early ovarian cyst development to promote the mitotic/meiotic switch, the dramatic growth of egg chambers later in oogenesis is a metabolically expensive process that is predicted to require high TORC1 activity. The current data indicate that the GATOR2 components Mio and Seh1 function to oppose the TORC1-inhibitory activity of the GATOR1 complex in the female germ line. In mio and seh1 mutants, TORC1 activity is constitutively repressed in the germ line of developing egg chambers, resulting in the activation of catabolic metabolism and the blocking of meiotic progression and oocyte development and growth (Wei, 2014b).
Previous data indicate that Mio and Seh1 act very early in oogenesis soon after the formation of the 16-cell cyst. The mio and seh1 ovarian phenotypes can be rescued by depleting the GATOR1 components nprl2, nprl3, or iml1 in the female germ line or by raising baseline levels of TORC1 activity by disabling an alternative TORC1 inhibitory complex, TSC1/2. These data are consistent with the model that the failure to maintain the meiotic cycle and the oocyte fate in mio and seh1 mutants is a direct result of inappropriately low TORC1 activity in the female germ line brought on by the deregulation of the Iml1/GATOR1 complex (Wei, 2014b).
Notably, null alleles of both mio and seh1 are viable, with many somatic tissues exhibiting no apparent developmental abnormalities and only limited reductions in cell growth. Thus, although Mio and Seh1 are critical for the activation of TORC1 and the development of the female gamete, these proteins play a relatively small role in the development and growth of many somatic tissues under nutrient-replete conditions. Whether this small role reflects the fact that components of the Iml1/GATOR1 complex are expressed at low levels in some somatic cell types or that the complex is present but needs to be activated by a signal, such as nutrient stress or a developmental signaling pathway, remains to be elucidated (Wei, 2014b).
In the future it will be important to gain a fuller understanding of the potential environmental and developmental inputs that regulate the activity of the Iml1/GATOR1 and GATOR2 complexes in multicellular organisms. These studies will provide much-needed insight into the basic mechanisms by which both environmental and developmental signaling pathways interface with the metabolic machinery to influence cell growth and differentiation (Wei, 2014b).
Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. This study investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In meiotic prophase 1 'early zygotene' oocytes, synapsis is only observed at the centromeres. It was also found that nonhomologous centromeres are clustered during this process. In 'mid-zygotene' oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In 'late zygotene' oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. It is proposed that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers (Tanneti, 2011).
Drosophila pro-oocytes develop within 16-cell cysts that are
arranged in temporal order within the ovary. Each ovary
contains several germaria, where pairs of pro-oocytes begin
their development and enter prophase in region 2a and a single
oocyte is selected by region 3. Oocytes are defined
by the presence of the synaptonemal complex (SC), which is
detected by antibodies to the transverse element C(3)G (Page, 2001),
a coiled-coil protein similar to proteins in budding yeast
(ZIP1), C. elegans (SYP-1, SYP-2), and mammals (SYCP1)
(Page, 2004; Watts, 2011). Zygotene pro-oocytes were identified by their patchy
C(3)G staining, as opposed to the thread-like staining typical
of pachytene. Furthermore, by comparing the amount of
synapsis to the relative positions of the pro-oocytes in the
wild-type germarium, three stages of zygotene were defined (Tanneti, 2011).
First, early zygotene pro-oocytes have one or two patches of
C(3)G that colocalize with CID, a centromere-specific histone H3. These pro-oocytes reside in the earliest
(most anterior) part of region 2a, indicating that synapsis initiates
at the centromeres before any other sites. These results
were confirmed by comparing CID localization to histone
modifications specific for the heterochromatin or euchromatin. Because there are four pairs of centromeres, the observation that most wild-type pro-oocytes
have one or two CID foci indicates that nonhomologous
centromeres cluster in meiotic prophase, confirming
previous observations using electron microscopy (Tanneti, 2011).
Second, mid-zygotene pro-oocytes have the centromeric
C(3)G staining plus approximately six additional sites in the euchromatin. Finally,
late zygotene pro-oocytes contain many C(3)G foci but lack
the continuous threadlike pattern of pachytene. Surprisingly,
the mid-zygotene patches do not appear to get longer.
Instead, there are more patches in late zygotene, suggesting
that the progression from mid- to late zygotene involves the
establishment of new SC initiation sites rather than polymerization
from the small number of sites in mid-zygotene. It is
suggested that the noncentromeric C(3)G sites in mid-zygotene
represent the first euchromatic sites to initiate synapsis. This study provides evidence that the mid-zygotene sites have features in common with centromere synapsis sites but are mechanistically distinct and genetically separable from the additional synapsis initiation sites observed in late zygotene (Tanneti, 2011).
C(2)M is a lateral element component and is a member of the Kleisen family that includes Rec8 and Rad21 homologs (Schleiffer, 2003). In wild-type, C(2)M colocalizes with C(3)G in most locations except at the centromeres. In
females lacking C(2)M, the first two stages of zygotene
appear to occur normally. Early zygotene pro-oocytes exhibit one or two foci of CID that colocalize with C(3)G, showing that C(2)M is not required for
centromere clustering or centromere synapsis. These results
confirm previous observations (Khetani, 2007) that C(2)M is not required
for centromere clustering in pachytene oocytes and are
consistent with the observation that C(2)M does not localize
to the centromeric regions. Early zygotene
in c(2)M mutants is followed by cysts with several
patches of euchromatic C(3)G staining that resemble wildtype
cells in mid-zygotene. Synapsis in a c(2)M mutant does not, however, progress beyond this point. Examination of histone modifications
in c(2)M mutants confirmed that synapsis is
blocked in mid-zygotene with a small number of euchromatin
initiation sites. Based on the similarities between wild-type
mid-zygotene and c(2)M mutants, it is suggested that synapsis
initiates in a c(2)M-independent manner at a small number
of specialized sites on the chromosomes, which include
approximately six euchromatic sites and the centromeres, and that C(2)M is required for additional initiation sites typical of late zygotene (Tanneti, 2011).
There is a striking similarity between the number of euchromatic
synapsis initiation sites (~6) during mid-zygotene and
the number of crossovers in Drosophila females. In
order to determine the relationship between SC initiation sites
and double-strand break (DSB) formation, c(2)M mutant
oocytes were stained for C(3)G and γ-H2AV.
DSBs in a c(2)M mutant are usually associated with a patch
of C(3)G staining (55/56 γ-H2AV foci were touching or overlapped
a patch of C(3)G). This experiment was also performed in
an okr mutant background (okr encodes the Drosophila homolog of Rad54) where the DSBs are not repaired and
γ-H2AV staining accumulates, allowing all DSBs to be counted. Most of the γ-H2AV foci in okr c(2)M mutant
germaria colocalized with a patch of C(3)G, suggesting
that the initiation of SC and recombination usually occur within
the same region in c(2)M mutants. Indeed, MEI-P22, a protein required for DSB formation, also colocalizes with the SC in
c(2)M mutant oocytes. It should be noted
that previous observations showed that DSB formation is
partially dependent on the SC. Indeed, the number of
γ-H2AV foci in the okr c(2)M double mutant in region 3 oocytes
was reduced compared to a okr single mutant. Overall, these results suggest that the SC, or a factor which stimulates SC formation, promotes
recruitment of proteins required for DSB formation (Tanneti, 2011).
To investigate whether there is a connection between early
SC initiation events and meiotic recombination, double
mutants with c(2)M were constructed. Unlike wild-type, where
γ-H2AV foci are not observed until pachytene, the block
in synapsis observed in c(2)M mutants allowed examination of
the relationship between SC initiation and DSB formation. By
double staining with CID, it was found that eliminating meiotic
DSBs with a mei-W68 mutation did not prevent formation of
either the centromere and euchromatic SC in a c(2)M mutant. The small decrease in the number of euchromatic SC sites in the c(2)M mei-W68
double mutant may indicate that the number of initiation sites
is sensitive to DSB formation. Furthermore, SC initiation is not
grossly affected by a reduction in crossing over (mei-218), an increase in crossing over (TM6), or a defect in DSB repair (okr). DSBs do not occur in the heterochromatin; thus, it is not surprising that centromere SC is independent of DSB formation. However, these results show that the initiation of euchromatic synapsis at mid zygotene does not depend on DSBs or crossovers (Tanneti, 2011).
Because DSBs or recombination are not required for synapsis in wild-type or c(2)M mutants, tests were performed to see whether structural components of the meiotic chromosomes regulate SC initiation. ORD is a meiosis-specific protein required for cohesion and crossover formation that may be a component of the SC
lateral elements. Although previous studies have shown that ord mutant oocytes generate threads of C(3)G staining that resemble pachytene, the effect of ord on zygotene progression has not been previously examined (Tanneti, 2011).
Consistent with previous results, this study found that centromere
clustering is defective and the association of
SC proteins with the centromeres is disrupted in ord mutant
oocytes. Furthermore, zygotene appeared abnormal;
rather than observing centromeric and euchromatic SC
initiation sites typical of mid-zygotene in early region 2a, it was
found that many ord mutant pro-oocytes with C(3)G staining only
around the nuclear DNA. Of the 108 pro-oocytes examined
in five germaria, 36 (33%) had no nuclear C(3)G. The remaining
pro-oocytes [72, (67%)] either had a number of C(3)G patches
that was more typical of late zygotene, usually in region 2a, or were in pachytene. It is concluded that the centromeric and euchromatic synapsis sites typical
of early and mid zygotene are absent in ord mutants, suggesting
that, in the absence of ORD, synapsis does not initiate normally (Tanneti, 2011).
Because ord mutants do eventually form threads of SC, it
was difficult to be sure that SC initiation was defective. To
test whether ord has a role in mid-zygotene synapsis, tests were performed to see whether the euchromatic patches of C(3)G in a c(2)M
mutant depend on ord. Even though both
single mutants exhibit at least some SC formation, most of the C(3)G staining in the c(2)M ord double mutant surrounded the DNA and within the nucleus.
This nonchromosomal C(3)G localization in the c(2)M ord double mutant was much more pronounced than in the ord single mutant. In
addition, C(3)G-staining ring-like structures were observed similar to what has been reported in some c(3)G missense mutants. All the nonchromosomal C(3)G staining may be due to polycomplex formation. c(2)M ord double mutant pro-oocytes were identified by the prominent C(3)G around the DNA, and the number of C(3)G patches on the chromosomes was found to be drastically reduced compared to wild-type zygotene or either single mutant (Tanneti, 2011).
These results demonstrate that ord is required
for the centromeric and euchromatic synapsis
sites observed in c(2)M mutants. Conversely,
C(2)M is required for the threadlike synapsis
observed in ord mutants. The synergistic
phenotype of the double mutant suggests that there are two
types of synapsis initiation - one depends on ORD (early and mid-zygotene) and the other depends on C(2)M (late zygotene) - and that these are independent events. In the absence of both types of synapsis initiation, C(3)G cannot load onto the chromosomes and accumulates in polycomplexes (Tanneti, 2011).
Like other Kleisin family members, C(2)M has been shown to
physically interact with the cohesin subunit SMC3 (Heidmann, 2004). To
determine whether C(2)M localization depends on an interaction
with cohesin, oocytes lacking SMC1 and
SMC3 were examined. To examine oocytes lacking SMC3 (encoded by cap), the recently developed short hairpin RNA (shRNA)
resource, which allows RNA interference (RNAi) knockdown
of gene expression in the Drosophila female germline, was used. Both the chromosomal
localization of C(3)G and C(2)M were absent when cap shRNA was expressed in the germline. Furthermore, SMC1 staining was eliminated, suggesting that the RNAi was effective at knocking out SMC3 function. Like the c(2)M ord double mutant, most C(3)G staining accumulated around the periphery of the DNA,
suggesting that the function of SMC3 in synapsis occurs
through at least two independent interactions with C(2)M and
ORD. Unlike the c(2)M ord double mutant, however, it was not possible to distinguish the pro-oocytes from the nurse cells because C(3)G staining was evenly distributed among the cells in each germarium cyst. Importantly, oocyte selection was not perturbed because one cell in each cyst accumulated ORB
protein, a cytoplasmic marker for the oocyte. Thus, the loss of SMC3 may have a more severe phenotype than the c(2)M ord double mutant (Tanneti, 2011).
These results were confirmed with the analysis of SMC1 mutant germline clones. As with cap RNAi, there was an absence of nuclear C(2)M and C(3)G threads in oocytes lacking SMC1, indicating a complete block in synapsis. Also similar to cap RNAi, the accumulation of ORB in one cell indicated that an oocyte was established. The only difference compared to cap RNAi was that there was much less C(3)G staining around the periphery of the DNA. It is not known whether this minor difference is due to the different methods (RNAi versus germline clone) or distinct functions of the two SMC proteins. Nevertheless, the results of these two experiments demonstrate that SMC1 and SMC3 are required for synapsis (Tanneti, 2011).
It is concluded that synapsis initiation during zygotene in
Drosophila females occurs in three stages. In early zygotene, the centromeres are the first sites to accumulate the transverse filament protein C(3)G. Indeed, cohesion proteins SMC1, SMC3, and ORD are detected at the centromeres before meiotic prophase (prior to or during premeiotic S phase), which could explain why synapsis is first observed at the centromeres. Interestingly, the SC also
forms first at the centromeres in budding yeast and depends on cohesion proteins. In mid-zygotene, synapsis initiates at a small number of euchromatic sites. These first two steps depend on the ORD protein. Finally, in late zygotene, synapsis initiates at a larger number of euchromatic sites. This stage requires C(2)M and appears to occur through a new set of initiation events rather than extending synapsis, or 'zipping up,' from the mid-zygotene initiation sites. Indeed, the synapsis initiation events in mid and late zygotene are independent and genetically separable, supporting a model where synapsis occurs through two independent waves of initiation events. In the absence of ORD, early and mid-zygotene synapsis events are skipped and the late zygotene initiation events occur with normal kinetics. This is not without
consequence, however, because at the electron microscopy
level, this synapsis is abnormal and tripartite SC is not visible. Both waves of synapsis initiation depend on the cohesin proteins SMC1 and SMC3, which may interact independently with C(2)M and ORD (Tanneti, 2011).
In addition to its role in centromere synapsis, ORD and the
SMC proteins are required for the pairing and clustering of
centromeres, whereas the SC components C(2)M or C(3)G
are not. Thus, cohesion proteins may be able to function in
a pairing role independent of DSBs, as Rec8 does in budding
yeast for centromere coupling. It is suggested that the first
euchromatic sites to initiate SC assembly in Drosophila are
in regions where cohesion proteins are most abundant. This
model is attractive because it provides a mechanism for SC
initiation in the absence of DSBs. Interestingly, the number
of euchromatic initiation sites in mid-zygotene or in c(2)M
mutants approximates the number of crossovers in the
genome. Not only do these mid-zygotene sites depend on ORD, but in ord mutants, crossing over is reduced to less than 10% of wild-type, even though DSBs occur normally. It is suggested that the reduction in crossing over in
ord mutants is due to the absence of the synapsis initiation
sites at mid-zygotene. Whether the synapsis initiation sites
actually correspond to crossover sites awaits further study (Tanneti, 2011).
ORD may have a function similar to yeast Rec8 because it is required
for synapsis at the centromeres and a subset of euchromatic
sites. Interestingly, the findings with C(2)M, which is not an
ortholog of Rec8, are also probably relevant to other species.
Several recent studies have revealed Non-Rec8 Kleisin
homologs in mouse and C. elegans (COH-3 and COH-). These parallels between the synapsis pathway in flies and that of organisms that depend on DSBs
for synapsis could reflect the existence of a conserved underlying
mechanism of synapsis. If synapsis initiation sites can
be marked prior to DSB formation in a process involving cohesion
proteins, and if proteins like Zip3 can be recruited in the
absence of DSBs, as is true in C. elegans and likely in
Drosophila, the timing of the DSB then becomes less of a determining
factor in the process of synapsis (Tanneti, 2011).
In prophase of the first meiotic division, chromatin forms a compact spherical cluster called the karyosome within the enlarged oocyte nucleus in Drosophila melanogaster. Similar clustering of chromatin has been widely observed in oocytes in many species including humans. To identify genes involved in karyosome formation, a large-scale cytological screen was carried out using Drosophila melanogaster oocytes. This screen comprised 3916 genes expressed in ovaries, of which 106 genes triggered reproducible karyosome defects upon knockdown. The karyosome defects in 24 out of these 106 genes resulted from activation of the meiotic recombination checkpoint, suggesting possible roles in DNA repair or piRNA processing. The other genes identified in this screen include genes with functions linked to chromatin, nuclear envelope, and actin. It was also found that silencing of genes with mitochondrial functions, including electron transport chain components, induced a distinct karyosome defect typically with de-clustered chromosomes located close to the nuclear envelope. Furthermore, mitochondrial dysfunction not only impairs karyosome formation and maintenance, but also delays synaptonemal complex disassembly in cells not destined to become the oocyte. These karyosome defects do not appear to be mediated by apoptosis. This large-scale unbiased study uncovered a set of genes required for karyosome formation and revealed a new link between mitochondrial dysfunction and chromatin organization in oocytes (Nieken, 2023).
Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. This study discovered that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which were named Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation (Martin, 2022).
During Drosophila oogenesis, efficient RiBi is required in the germline for proper GSC cytokinesis and differentiation. The outstanding questions that needed to be addressed were: (1) Why does disrupted RiBi impair GSC abscission? And (2) How does the GSC monitor and couple RiBi to differentiation? The results suggest that a germline RiBi defect stalls the cell cycle, resulting a loss of differentiation and the formation of stem cysts. It was discovered that proper RiBi is monitored through a translation control module that allows for co-regulation of RPs and a p53 repressor. Ais, Ath, and Pths support RiBi and allowing for translation of a p53 repressor, preventing p53 stabilization, cell-cycle arrest, and loss of stem cell differentiation (Martin, 2022).
The developmental upregulation of p53 during GSC differentiation concomitant with reduced RiBi parallels observations in disease states, such as ribosomopathies. This study found that p53 levels in GSCs are regulated by the conserved p53 regulator Non1. Although Non1 has been shown to directly interact with p53, how it regulates p53 levels in both humans and Drosophila is not known (Martin, 2022).
TOP-containing mRNAs are known to be coregulated to coordinate ribosome production in response to environmental cues. Surprisingly, the observation that loss of ais reduces translation, albeit indirectly via regulation of RiBi, of a cohort of TOP-containing mRNAs, including Non1, suggests that the TOP motif also sensitizes their translation to lowered levels of RiBi. This notion is supported by TOP reporter assays demonstrating that reduced translation upon loss of ais requires the TOP motif. It is hypothesized that limiting TOP mRNA translation lowers RP production to maintain a balance with reduced rRNA production. This feedback mechanism would prevent the production of excess RPs that cannot be integrated into ribosomes and the ensuing harmful aggregates (Martin, 2022).
The translation and stability of TOP-containing mRNAs are mediated by Larp1 and its phosphorylation. This study found that perturbing rRNA production and thus RiBi, without directly targeting RPs, also results in dysregulation of TOP mRNAs. The data show that Drosophila Larp binds the RpL30 and Non1 5' UTR in a TOP-dependent manner in vitro and to 97% of the translation targets were identified in vivo. Together, these data suggest that rRNA production regulates TOP mRNAs via Larp albeit indirectly. Furthermore, the cytokinesis defect caused by OE of Larp-DM15 in the germline suggests that Larp regulation could maintain the homeostasis of RiBi by balancing the expression of RP production with the rate of other aspects of RiBi, such as rRNA processing, during development (Martin, 2022).
Ribosomopathies arise from RiBi defects. The underlying mechanisms of tissue specificity remain unresolved. This study demonstrates that loss of proteins involved in rRNA processing lead to cell-cycle arrest. Given that Drosophila GSCs undergo an atypical cell cycle as a normal part of their development it may be that this underlying cellular program in the germline leads to the tissue-specific phenotype of stem-cyst formation. This model implies that other tissues would likewise exhibit tissue-specific manifestations of ribosomopathies due to their underlying cell state. The data suggest two other sources of potential tissue specificity: (1) tissues express different cohorts of mRNAs, such as Non1, which are sensitive to ribosome levels (2). p53 activation, as previously described, is differentially tolerated in different tissues. Together, these mechanisms could begin to explain the tissue-specific nature of ribosomopathies and their link to differentiation (Martin, 2022).
The exact processing steps that Ais, Ath, and Pths promote in Drosophila RiBi remain unknown; it is hypothesized that the processing step they act on the rRNA would be similar to what has been reported in yeast and mammals . Lack of a full rescue from ais, ath, and pths GKD in p53 mutants suggest that multiple genes likely influence the cell-cycle arrest. Finally, it is possible that the roles of Ais, Ath, and Pths in indirectly promoting Non1 translation does not represent a general effect of RiBi defects and is specific to these three proteins. However, this is thought unlikely as nearly all genes involved in RiBi outside of RPs share the same phenotype when depleted during Drosophila oogenesis (Martin, 2022).
In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see Verthandi) or cohesin regulators are knocked down after meiotic S phase. This study used two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase (Haseeb, 2023).
The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. This manuscript showa that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, these results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis (Feijao, 2023).
The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, a strategy was developed to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. An interaction between Borealin and the chromatin was found to be crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. It is proposed that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation (Wang, 2021).
Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, the origin of oocyte and germ plasm-associated mitochondria was studied during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton since it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. These findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support the hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles (Cox, 2003).
Drosophila oocytes contain a typical Balbiani body
at the time follicles form in region 3 of the germarium. In a wide range of
animal species, including Xenopus, chick, mouse and human, young oocytes at a similar developmental stage display these distinctive aggregates of mitochondria and other organelles near their germinal vesicles. In a typical Balbiani body, centrioles and associated cytoplasm are surrounded by a ring of Golgi bodies and encased in a large mass of mitochondria. As the oocyte grows, the mitochondria first spread around the nuclear periphery and later disperse throughout the oocyte cytoplasm (Cox, 2003).
Drosophila Balbiani bodies, like those described in other species,
contain clustered mitochondria, Golgi vesicles and centrioles.
Moreover, as young follicles develop from stage 1-6, the mitochondria move
around the germinal vesicle and disperse after microtubules re-organize in
stage 7 (Cox, 2003).
The studies reported here provide new insight into the origin of Balbiani
bodies. Drosophila Balbiani bodies do not arise de novo within
oocytes, but are built by the transport of organelles from neighboring cells
within interconnected germline cysts. These experiments make clear that many
components of oocyte cytoplasm arise in this manner (Cox, 2003).
Virtually all of the newly formed
mitochondria in oocytes are derived from the Balbiani body. The great majority
are transported from other cystocytes along the fusome but 1/16th or more
might simply originate in the oocyte. Like oocyte determination itself,
Balbiani body formation depends on the fundamental cyst polarity
manifested in the fusome. Arising in embryonic germ cells, the
fusome builds up a framework of cyst polarity during the cystocyte divisions. Fusome polarity probably acts directly to control centriole
migration and the meiotic gradient, and acts indirectly to differentiate and maintain the oocyte by regulating the microtubule cytoskeleton.
Deciphering the molecular mechanisms that define fusome polarity and allow the
fusome to control microtubule organization remains a central issue for
understanding Balbiani body formation and oocyte development (Cox, 2003).
Oocytes develop from germline cysts or syncytia in diverse species so Balbiani bodies may arise through intercellular transport
in a wide range of organisms besides Drosophila. In both
Xenopus and the mouse, mitochondrial clouds present within
interconnected germ cells are thought to be precursors to the Balbiani bodies
that arise shortly after the cysts break down and form primordial follicles.
In Drosophila, the large chunk of fusome at the anterior of the early
stage 1 oocyte contains clustered centrioles and is likely to act as a
microtubule-organizing center. It may attract and retain mitochondria, Golgi and
localized macromolecules as they enter the oocyte, thereby creating the
Balbiani body. Xenopus Balbiani bodies may arise in a similar fashion
as they have a similar organization consisting of a spectrin-rich zone,
mitochondria, Golgi and the Metro region containing RNAs in transit. However,
there has been insufficient study of the Xenopus larval ovary to
identify a fusome or some other material with microtubule organizing properties that might play an analogous role. In most other systems whose Balbiani bodies share the same basic structure in young oocytes, very little is known about their origin during earlier stages of germ cell development (Cox, 2003).
The Balbiani bodies in many species contain structures resembling germinal
granules. In Xenopus, these granules are found in a region containing
specific RNAs that are also destined to be localized in the egg and
incorporated in germ cells. Consequently, the Balbiani body has been proposed
to function as a messenger transport organizer (METRO) that organizes and
mediates the delivery of RNAs and germinal granules to the vegetal pole of the
egg. Specific elements have been mapped in the 3' UTR of the Xcat2 mRNA that are sufficient for localization to the Balbiani body or
to the germinal granules themselves (Cox, 2003).
The Drosophila Balbiani body may play a
related role. oskar RNA, a key component that is capable of inducing
germ plasm formation, is associated with the posterior segment of the
Balbiani body in early stage 1 oocytes, much as Xcat2 is localized in
the Xenopus Balbiani body. A few hours later, towards the end of
stage 1, osk RNA moves to the oocyte posterior along with the other
Balbiani-associated RNAs and proteins that have been studied, presumably in response to the shift in microtubule polarity that occurs at this time. Thus, at least some molecules that participate in germ plasm assembly associate with the
Balbiani body in early Xenopus and Drosophila oocytes (Cox, 2003).
Drosophila RNAs that become associated with the
Balbiani body, like organelles, first interact with the fusome during early
stages of cyst development. However, there are significant differences in
these fusome interactions with RNAs and organelles that probably reflect different molecular mechanisms
of delivery to the Balbiani body. Organelles associate next to the fusome
along much of its length and subsequently move toward the center, in concert
with microtubule minus ends. By contrast, the RNAs associate with one or a few
cells at the center of the fusome from the earliest stages they could be
detected, and are located within it, as well as nearby. These observations
suggest that localized RNAs may read the fusome polarity directly, and need
not rely on changes in microtubule organizing activity to get to the oocyte or
be stabilized within it (Cox, 2003).
Potentially significant differences exist in the role of RNA transport
played by the Drosophila and Xenopus Balbiani bodies. The
Drosophila Balbiani body associates with germ plasm RNAs for only
5-10 hours during early stage 1. By contrast, Xenopus Balbiani bodies
associate throughout stage 1 of oogenesis, a process requiring many days, with
at least 11 RNAs. When the RNAs leave the Drosophila Balbiani body,
mitochondria mostly remain behind, only to follow much later in oogenesis. By
contrast, in Xenopus, both mRNAs and mitochondria are reported to
proceed together to the vegetal pole. These differences may simply reflect differences in the timing of cytoskeletal remodeling that control these events. Moreover, the observation that a small subset of mitochondria recognized by COXI
antisera do translocate with the RNAs in stage 1 indicates that certain
Drosophila mitochondria may follow a Xenopus-like pattern. However, it remains possible that RNAs in transit to the oocyte posterior may simply pass through the Balbiani body without being affected in any way (Cox, 2003).
Sponge-like structures have been described in the cytoplasm of stage
4-10 nurse cells that are associated with Exu protein, RNA, and (frequently)
mitochondria and nuage. It has been proposed that these structures are analogous to classical Balbiani bodies and that they mediate transport of localized
transcripts such as bicoid RNA. The current results suggest that the ooctye contains a true Balbiani body much earlier -- in stage 1 follicles. The sponge bodies more likely represent transport complexes organized at the
surface of nurse cell nuclei that subsequently move through the follicle and
into the ooctye. However, there may be structural and molecular similarities
between nurse cell transport complexes and those mediating transport out of
the Balbiani body (Cox, 2003).
These studies provide further evidence that the ring canals that join the
cystocytes play an important role in regulating Balbiani body formation.
Mitochondria appear to first enter the oocyte when fusome segments within the
adjoining ring canals break apart, unplugging the channels. Subsequently, a
novel mechanism blocks further mitochondrial passage through these canals,
because large backups of mitochondria are observed outside each oocyte ring
canal in young oocytes and a lack of mitochondrial movement into
the oocyte has been documented in movies. Mitochondria do not accumulate in the same manner around the ring canals that join nurse cells, but are spread throughout the cell and in the nuclear periphery. This behavior has the effect of limiting the mitochondrial genotypes within the oocyte to those found in Balbiani body mitochondria until well after mitochondria have begun to associate with the
germ plasm at the oocyte posterior pole. Despite the importance of these
regulatory steps, little is known about how movement through ring
canals is controlled (Cox, 2003).
These studies suggest that centrioles, mitochondria, Golgi, RNAs and other
key components of oocyte cytoplasm are added to the Drosophila oocyte
by a special mechanism that may have been widely conserved in evolution. It is
remarkable that in the oocyte, the lone cell that will contribute cytoplasm
for the next generation of organisms, many fundamental components of cytoplasm
do not arise by random partitioning among daughter cells. Rather, an elaborate
mechanism is used to transport materials from multiple cells and maintain them
in a large aggregate for an extended period of time. It is possible that
Balbiani bodies do not play a specific role in ooctye development, but
represent a byproduct of the unusual centrosome behavior in these cells.
However, an alternative hypothesis is favored. One of the potentially most interesting reasons that oocyte organelles might be delivered en mass via the fusome would be to increase organelle fitness. Mitochondrial DNAs are known to accumulate mutations that have frequently been postulated to affect the aging of cells and tissues. If only mitochondria with functional genomes are able to
associate with the fusome and move into the oocyte, damaged genomes might be
weeded out when they still represent a small fraction of the total. Such a
system would be far more efficient than eliminating defective genomes by
inducing the apoptosis of entire germ cells. A purifying mechanism based on
organelle selection might be particularly important in organisms that need to
produce eggs with a high average viability, or that must support long
intergenerational life spans (Cox, 2003).
Several other observations may also be explained by the need to eliminate
defective mitochondrial genomes. The exclusion of nurse cell mitochondria from
passing through the oocyte ring canals prior to dumping would ensure that only
the 'selected' mitochondria in the Balbiani body populate the germ plasm.
Mitochondria may break up into small, nearly round, organelles during this
period so that each will contain a single genome whose fitness can be tested.
The cytoplasmic streaming of the ooctye may serve to mix the two populations
of organelles so each somatic cell type inherits at least some of the selected
mitochondrial population. Finally, a requirement for translation on
mitochondrial ribosomes in the early embryonic germ plasm might serve as a
concluding selective step to ensure that viable germ cells are well supplied
with intact mitochondrial genomes. If female germ cells do possess mechanisms
to remove defective mitochondria, they would probably have contributed to the
evolutionary conservation of germ line cysts and Balbiani bodies (Cox, 2003).
Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. This easy-to-score eggshell phenotype was used in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. This study describes the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba (CG8174), the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. These findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis (Barbosa, 2007),
In this study, a clonal screen was used to identify genes regulating meiotic progression in Drosophila. Instead of testing directly for defects in meiosis, an easy-to-score eggshell phenotype was used that is produced when the levels or activity of the morphogen Grk are affected. This allowed an efficient screen of a large number of mutant lines and identification of germ-line-specific genes as well as genes with essential functions. The number of new genes identified is likely less than the total number of 2R genes required for Grk synthesis and function since mutations were discarded that blocked oogenesis. Of the eight genes described in this study, five show meiotic phenotypes. dPds5, nds, and mtc delay meiotic restriction to the oocyte, although only dPds5 and nds genetically interact with mei-W68 and mei-41, respectively. trin and blv affect the morphology of the karyosome in spite of normal timing in meiotic restriction. This confirms the effectiveness of the screening method for meiotic genes. Genetic and developmental analysis of the newly identified genes provides evidence for new regulatory steps in a network that coordinates Drosophila meiosis and oocyte development (Barbosa, 2007),
One complementation group, cohiba, identifies the Drosophila homolog of Pds5p in Schizosaccharomyces pombe, Spo76 in Sordaria macrospore, and BimD in Aspergillus nidulans, which have been found associated with the cohesion complex of mitotic and meiotic chromosomes. Depletion of Pds5 affects not only cohesion but also condensation in meiotic prophase. The unique 'open chromatin' karyosome defect observed in dPds5cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5cohiba phenotype is suppressed by Spo11 (mei-W68) mutations defective in DSB formation. This suggests that dPds5 is necessary to maintain the structure of the meiotic chromosomes after DSBs are induced. However, in contrast to known DSB repair genes, the meiotic delay and oocyte patterning defects of dPdscohiba mutants are not due to activation of ATR/Mei-41-dependent checkpoint. One possibility is that the ATR downstream effector kinase dChk2 is activated via an alternative pathway, such as the Drosophila ataxia-telangiectasia mutated (ATM) homolog, which indeed activates dChk2 in the early embryo independently of ATR. Alternatively, dPdscohiba mutants may activate a checkpoint that measures cohesion rather than DSB breaks. The only other cohesion protein characterized in Drosophila is the product of the orientation disruptor (ord). ORD plays a role in early prophase I by maintaining synaptic chromosomes and allowing interhomolog recombination. More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair. However, in contrast to dPds5 mutants, karyosome morphology is normal in ord mutants, and an eggshell polarity phenotype has not been reported. Although required for chromatid cohesion, dPds5 and ORD might play complementary roles in SC dynamics: ORD may stabilize the SC in the oocyte, whereas dPds5 may be required for the disassembly of synapses as one of the pro-oocytes regresses from meiosis (Barbosa, 2007),
The screen identified mutations in montecristo (mtc) that affect the restriction of meiosis to the oocyte. It has been proposed that this delay reflects the activation of the ATR/Mei-41 checkpoint pathway. Similar to dPds5, Mtc may control the regression from pachytene in those cyst cells that will not adopt the oocyte fate. The delayed meiotic restriction observed in mtc mutants occurs, however, independently of DSB formation or Mei-41 checkpoint activation. Mtc contains a Tudor domain. In other Tudor-domain proteins, this domain has been shown to interact with methylated target proteins. Identification of specific Mtc targets may clarify its role in meiotic restriction and oocyte patterning (Barbosa, 2007),
A particularly intriguing and novel phenotype is uncovered by mutations in indios (nds). By delaying meiotic restriction and activating Mei-41 without affecting the karyosome morphology, nds mutants separate checkpoint activation leading to Grk decrease from checkpoint activation controlling karyosome compaction. The nds phenotype also occurs independently of DSBs, suggesting that the trigger that leads Nds to trigger checkpoint activation is not DNA breaks. The fact that nds mutants are extremely sensitive to Mei-41 dosage further suggests that Nds activity may specifically control a branch of the Mei-41 checkpoint regulating Grk activity. In contrast to nds, trin mutants do not delay meiotic restriction and show defects in the karyosome in spite of normal Grk levels. Like mutants in src64B and tec29, which show a similar phenotype, Trin may mediate chromatin remodeling in the oocyte by regulating the actin cytoskeleton. In this context, the DV phenotype of eggs from trin mutants may be an indirect effect due to defects in actin cytoskeleton function. The production of collapsed eggs by trin mutant germ-line clones is consistent with this idea (Barbosa, 2007),
Finally, blv mutants show striking similarity to vas mutants with respect to lack of sensitivity to DSB formation, no evident delays of meiotic restriction, or karyosome and Grk phenotypes. Blv may thus act downstream or independent of the Mei41/ATR checkpoint, and its further characterization may help to understand the effector side of the meiotic checkpoint pathway (Barbosa, 2007),
Previous knowledge pointed to Drosophila meiosis as a linear progression of events from homologous chromosome pairing and recombination to meiotic restriction, karyosome formation, and eggshell patterning, with DSB repair as the main checkpoint linking meiosis to Grk signaling. By uncoupling some of these events, this study suggests the existence of a more complex network that links the surveillance of meiotic progression to oocyte patterning (Barbosa, 2007),
The onset of development is marked by two major, posttranscriptionally controlled, events: oocytematuration (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, proteome remodeling has been described during Drosophila egg activation. This study describes quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. Muskelin, found to be up-regulated during oocyte maturation, was shown to be required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. This study found down-regulation of this class of proteins to be mediated partially by APC/CORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C) (Kronja, 2014).
In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being 'movers' to 'fertilizers'. Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. Within the female D. melanogaster, both gamete maturation and sperm storage are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization (Qazi, 2003).
Mating initiates a series of events within the female reproductive tract, including ovulation and sperm storage. Ovulation and sperm storage occur in different regions of the female reproductive tract, but are coordinated for a connected fate: the fertilization of an egg. At the anterior of the female Drosophila reproductive tract are two ovaries, each composed of 10-20 ovarioles. The ovarioles are held together by a peritoneal sheath, containing a network of fine, branching muscle fibers. The base (proximal end) of each ovariole forms a small duct or pedicel. The pedicels of all the ovarioles in an ovary unite to form a calyx; each calyx opens into a lateral oviduct. The lateral oviducts fuse into a common oviduct that, more posteriorly, enlarges to form the uterus. The wall of the oviducts consists of epithelium surrounded by circular muscles that is intensively innervated. The uterus is a heavily muscularized and innervated structure that receives sperm during mating and also holds the egg in position for fertilization. At the anterior end of the uterus are the three sperm storage organs: a single seminal receptacle and the paired spermathecae, as well as the paired spermathecal glands (also called parovaria or female accessory glands). At its posterior end, the uterus narrows forming the vagina that exits the female reproductive tract. The distal end of the vagina, called the gonopore, serves for the discharge of eggs (Qazi, 2003 and references therein).
In many organisms, interaction between the gametes triggers a series of cellular responses in the egg ('egg activation') required to initiate embryonic development. Drosophila eggs also are triggered to initiate a suite of cellular responses analogous to those of activation in other organisms (relief of arrest in meiosis, ultimately leading to completion of meiosis, changes in egg coverings, and initiation of translation) in a process that has therefore also been called activation. This semantic convergence masks a distinct mechanistic difference in the trigger for activation in Drosophila and at least one other insect relative to that in echinoderms, nematodes, and vertebrates: egg activation in Drosophila and other insects initiates independent of sperm entry, perhaps not surprising in an Order that includes species in which haploid males can develop from unfertilized eggs. In Drosophila, changes in the egg envelope's permeability, one feature of activation, initiate during ovulation, even while most of the egg is still within the ovary (Heifetz, 2001b). The egg's covering becomes progressively more impermeable to small molecules as the egg proceeds down the oviduct and the process is complete by the time the egg arrives at the uterus. Cross-linking of vitelline membrane protein sV23 also increases progressively as the egg moves through the oviduct and the uterus. Ovulation also triggers meiosis to resume before the egg reaches the uterus (Heifetz, 2001b). Thus, ovulation causes changes in the egg that prepare it for subsequent embryogenesis, should fertilization occur in the uterus, including coordination between cell cycle status of the egg and the sperm nuclei. The third feature of activation, resumption of translation, also initiates without fertilization in Drosophila, though it is not yet known if it too is triggered by ovulation (Qazi, 2003 and references therein).
Drosophila females produce up to two final-stage (stage 14) oocytes in each ovariole daily (as many as 80 oocytes/day. Several control points and feedback mechanisms regulate the production of mature oocytes. If mature oocytes are not ovulated, each ovariole accumulates two or three late-stage oocytes. This blocks the maturation of additional oocytes. Mating, and specifically sperm and some male accessory gland proteins [e.g., Acp26Aa (ovulin)], induces ovulation of mature oocytes (Heifetz, 2000 and Heifetz, 2001b) that, in turn, contribute to stimulating oogenic progression via a feedback mechanism. Thus, ovulating females produce high numbers of mature oocytes and deposit high numbers of fertilized eggs. The feedback mechanism by which seminal fluid, sperm, and the act of mating itself increase oogenic progression rate is not known (Qazi, 2003 and references therein).
Drosophila virgin females retain mature oocytes in their ovaries. Ovulation initiates within 1.5 h after mating (Heifetz, 2000) during a time when sperm are still being stored. Thus, the initial eggs ovulated are released potentially prior to full completion of sperm storage. The number of progeny per number of eggs laid (hatchability) immediately after matings between wild-type males and females is lower than at later time points (Chapman, 2001). This lower hatchability appears to reflect a lower efficiency of fertilization of the first eggs released. By visualizing the fertilizing sperm tail within wild-type eggs, it was found that the first eggs laid had a lower fertilization efficiency (ratio of unfertilized/fertilized eggs = 1.5 at 3-4 h post-mating) than those laid subsequently (0.6 at 48-49 h, Chapman, 2001). Two possible explanations for the low hatchability are suggested; regulated ovulation plays a critical role in each of these. (1) Ovulation allows coordination of oocyte release with the rate of sperm release from storage. The first eggs ovulated may reach the fertilization site before sperm are prepared and in position to be released from storage. Thus, lower numbers of those eggs would be fertilized, leading to lower hatchability. (2) Since unmated females can accumulate eggs for several days, it is possible that the older eggs are 'stale' and cannot be fertilized. By ovulating those eggs quickly, even before sperm are fully stored, the female clears out any stale eggs without wasting sperm (Chapman, 2001). Understanding the mechanism that coordinates oocyte release from the ovary and sperm release from the sperm storage organs will provide insights into the first steps of sperm/egg interactions that lead to successful fertilization (Qazi, 2003 and references therein).
In insects, production and laying of eggs requires five steps within the female reproductive tract: (1) oogenesis (the generation of oocytes within the ovary); (2) ovulation (release of an oocyte from the ovary into the oviducts); (3) movement of the egg down the oviducts; (4) fertilization of the egg when it has come to rest in the uterus, and (5) deposition of eggs onto the substratum. These steps are sequential and thus somewhat coupled. Therefore, assays of oocyte/egg progression sometimes measure several of these steps at once (Qazi, 2003).
Ovulation itself results from a series of processes occurring both sequentially and simultaneously within several ovarian microenvironments. The oocyte is released from its follicle, squeezed out of the ovary, and pushed through a lateral oviduct into the common oviduct, coming to rest in the uterus. Within each Drosophila ovariole, oocytes develop in a sequential fashion. Each ovariole contains a series of four to six egg chambers at stages in developmental progression (stages 1-14), with oogonia at the apex of the ovariole and the most mature oocytes (stage 14) at the base of the ovariole. In each egg chamber, the oocyte, connected to its sister cells (the nurse cells), is surrounded by a monolayer of somatic follicle cells. Follicle cells synthesize some of the yolk protein that will be deposited into the oocyte, as well as the proteins of the vitelline envelope and chorion that will cover and protect the oocytes. Specialized follicle cells at the anterior end of the egg chamber synthesize the micropyle, the site on the egg through which the sperm will enter. When the adult female fly ecloses, the base of the ovariole is still plugged with cells that grew out of the pedicel; this plug must be breeched to allow ovulation (Qazi, 2003 and references therein).
The mechanism by which oocytes are released from the Drosophila ovary is not known, but results in other insect systems suggest possible mechanisms. Insect ovulation involves two distinct steps: the opening of the oocyte follicle and of the intermost cells in the pedicel region, which releases the oocyte from the ovariole, and contraction of ovarioles, pedicels, and oviduct musculature, which moves the oocyte into the lateral oviducts. These contractions do not appear to involve sphincter(s), which are absent between the pedicels and the lateral oviducts in Drosophila. Ultrastructural studies of ovulation in the large aquatic beetle Dysticus marginalis (Coleoptera) show that the appearance of the first mature oocyte at the pedicel region triggers autolysis of the pedicel's innermost cells, opening the plug and releasing the mature oocyte from the ovariole. In addition, there are cells in the pedicel region that produce vacuoles whose contents might help the mature oocyte to glide into the oviduct. In the mosquito Culiseta inornata (Diptera), histological sections of whole ovaries also show that the pedicel is destroyed during ovulation. Thus, though little is known about the process of ovulation in Drosophila, it is likely that ovulation occurs, as in other insects, by cell degeneration in the pedicel region and massive muscle contractions at the base of the ovary and oviducts. These move the oocyte from the ovariole into and down the oviducts, where it eventually becomes lodged in the uterus. The posterior end of the oocyte leaves the ovary first. Thus, when the egg comes to rest in the uterus, its anterior end is 'up' with the egg's micropyle adjacent to the opening of the sperm storage organs, allowing for efficient fertilization (Qazi, 2003 and references therein).
In some insects, ovulation initiates only after mating. In Drosophila, however, ovulation occurs at a low level in adult females even without mating. Although Drosophila mature virgin females spontaneously ovulate at a very low rate (~1 egg/day), mating dramatically increases female ovulation rate (Heifetz, 2000). This effect of mating is rapid; an increase in ovulation is evident by 90 min post-mating (Heifetz, 2000). Some insects, such as walking sticks, ovulate one oocyte at a time and their ovarioles function alternately or in sequence, although in other insects, such as Orthoptera, all ovarioles ovulate simultaneously. It is not known which mechanism operates in Drosophila (Qazi, 2003 and references therein).
In Drosophila females, mating stimulates an increase in ovulation rate. Since ovulation is one step in the multistep process of egg laying, an assay was needed to distinguish this from the larger egg laying process. By measuring the progression of egg movement through the female reproductive tract, Heifetz (2000) showed that, by 90 min after mating, 90% of the females had at least one egg in their reproductive tract, and most of those were in the lateral oviduct. Ovulation rate increases with time after mating, such that by 6 h after mating, 30% of females have more than one egg in their reproductive tract. Seminal fluid components, Acps (accessory gland proteins), and sperm that are transferred to the female during mating play an important role in increasing female ovulation rate (Qazi, 2003 and references therein).
One seminal fluid protein, the prohormone-like Acp26Aa (also called 'ovulin') that shows sequence similarity to the Aplysia egg laying hormone, is essential for stimulating ovulation. The ovulation rate of females mated to males that lack Acp26Aa is 44% lower than the ovulation rate of mates of wild-type males (Heifetz, 2000). Acp26Aa's effects are evident between 1.5 and 6 h post-mating, and the protein is detectable in mated females for only a few hours post-mating. Since Acp26Aa stimulates ovulation shortly after mating, it is thought to act in 'clearing' the mature oocytes from the ovary, allowing for coordination of fresh oocyte release with sperm release and for increased oogenic progression rate (Chapman, 2001 and Heifetz, 2000). It is not yet known how Acp26Aa mediates oocyte release. Some Acp26Aa localizes at the base of the ovary (Heifetz, 2000), where it is processed into bioactive peptides. Some Acp26Aa enters the circulatory system. Thus, it is possible that Acp26Aa acts locally within the reproductive tract and/or via the neuroendocrine system to mediate contractile activity of the ovary and the oviduct musculature (Qazi, 2003 and references therein).
Another Acp, the 'sex peptide' Acp70A, also causes an increase in the number of eggs laid and, in one assay, the number of eggs in the uterus of dissected females. Since this Acp is known to increase oogenic progression, it is presently unknown whether the increased number of eggs seen in the uterus following Acp70A induction is simply a secondary indirect consequence of the increased production of oocytes, or is due to a separate direct effect on ovulation (Qazi, 2003 and references therein).
Drosophila males produce a peptide related to Acp70A in their ejaculatory ducts (Fan, 2000 and Saudan, 2002). Injection of this peptide, Dup99B, into unmated females can stimulate egg production. As with Acp70A, it is not presently known how Dup99B stimulates egg production, and therefore whether its effect on ovulation is direct or indirect (Qazi, 2003 and references therein).
In the absence of sperm transfer, oocyte development (from rapid yolk accumulation to vitelline membrane development and chorion deposition) and egg laying rates are lower than wild-type levels (50% and 33%-70% lower, respectively. Females that receive no sperm also begin to deposit eggs later than females mated to wild-type males (6 h vs 3 h; (Heifetz, 2001a). Moreover, even when sperm are transferred, if they are not stored, egg laying is low (<50% wild-type levels). Finally, when a female uses up all sperm from her mating, her egg laying rate decreases to virgin
levels. These results indicate that the transfer and storage of sperm is essential to elevate oogenesis and egg deposition rates and may also affect ovulation (Qazi, 2003 and references therein).
The presence of sperm in the reproductive tract may trigger changes in female fecundity by causing the release of female-derived activating substances or by releasing male-derived substances that have adhered to the sperm. Alternatively, the presence of many sperm could stimulate the female central nervous system via stretch receptors in the uterus or sperm storage organs (Qazi, 2003 and references therein).
Contraction of insect oviducts is under neural control. The Drosophila female reproductive tract is innervated by branches of the abdominal nerve center (AbTNv). Thus, it seems likely that neurotransmitters and/or neurohormones will regulate the contractile activity of the Drosophila ovary and oviduct musculature to cause the release of mature oocytes. Since the Drosophila female reproductive tract's epithelium has secretory characteristics, it is also likely that neurotransmitters or neurohormones released at the base of the ovary could affect the secretory activity of the epithelium, and thereby trigger the disintegration of the pedicel plug to release mature oocytes (Qazi, 2003 and references therein).
Although the identity of neurotransmitters, neuromodulators, or neurohormones that could mediate ovulation in Drosophila is unknown, findings in other insects point to likely candidates. In locusts (Locusta migratoria), for example, glutamate, octopamine, proctolin, and SchistoFLRFamide have been shown to mediate oviduct muscle contraction, aiding the movement of eggs. In other insects, such as the bed bug Rhodnius prolixus, the neurosecretory peptide myotropin mediates the ovarian contractile activity that releases mature oocytes. Myotropin is released from the corpus cardiacum in response to ecdysteroid levels in the female hemolymph (Qazi, 2003 and references therein).
In Drosophila, ovarian function and ovarian environment affect egg chamber development and therefore ovulation rate. Female age, available nutritional resources (e.g., yeast), and other environmental factors, such as circadian cues and humidity, affect egg production. For example, nutritional supplementation with yeast causes increased germ cell proliferation and decreased cell death of both early-stage and vitellogenic-stage oocytes. This results in increased egg production and egg deposition rates thereby (directly or indirectly) increasing ovulation rates. Although the mechanism of environmental interaction with ovulation physiology is still to be determined in Drosophila, again clues are available from other insects. For example, in the beetle Xyleborus ferrugineus, ecdysteroid titers are significantly higher in young fertile adult females than in aging females, suggesting that fewer eggs are produced, and hence ovulated in older females, due to declining ecdysteroid levels (Qazi, 2003 and references therein).
In Drosophila, several control points and feedback mechanisms regulate the production of mature oocytes, which affects female ovulation status. One such feedback prevents oocytes from entering the oviduct before completing oogenesis. Thus, a female will ovulate only if she has mature oocytes in her ovaries. Another control point in egg maturation is regulated by Acp70A. Acp70A stimulates oocytes to progress to later vitellogenic stages and thus override a control point in egg maturation between mid/late vitellogenic stages (stages 9/10). Thus, Acp70A can stimulate oogenic progression rate, allowing the female to ovulate at a high rate (Qazi, 2003 and references therein).
Sperm storage allows sperm from a given mating to be used long after the male and female have separated (~2 weeks), thus increasing female fertility. Of the ~4000 sperm transferred to a Drosophila female during mating between wild-type flies, ~80% are expelled from the uterus when the first egg is laid, and subsequent fertilizations rely on the 700-1000 sperm stored in the female. Females that receive normal quantities of sperm, but store few of them, produce few progeny (~10% wild-type levels). Compared with mammals and birds, whose efficiency of sperm use can be as low as 0.01% (reviewed in Neubaum and Wolfner, 1999b), Drosophila use sperm quite efficiently: of the 700-1000 sperm stored, ~400 are used to fertilize eggs (Qazi, 2003 and references therein).
Sperm storage potentially allows coordination of ovulation rate with sperm release from storage. This prevents gamete wastage, which would occur when gametes are released at different times and unable to unite successfully. The coordination of sperm release and ovulation could also decrease the incidence of polyspermy that also wastes eggs and sperm because it results in nonviable fertilizations. The controlled release of sperm from storage in Drosophila may be one reason why polyspermy is rare in this organism (less-than or equal to 1% (Qazi, 2003 and references therein).
Sperm storage allows females to retain sperm for more than one male within her reproductive tract. This provides 'fertility insurance,' should any of the males be infertile or genetically incompatible with the female. Storing sperm from multiple males has the potential additional advantage for females of generating progeny with a broader range of different genotypes. Finally, storing sperm from more than one male generates an opportunity for sperm competition and female sperm preference (Qazi, 2003 and references therein).
After mating, Drosophila females become unreceptive to courting males for several days (mating effect). Male seminal products such as Acp70A trigger the female's immediate reluctance to remate, but their action is short-term (<24 h). For the normal (approximately 1-1.5 week) depression of receptivity, females must also store sperm (sperm effect). The sperm effect on female receptivity may begin as early as 6-8 h postmating; it is clearly operating by 10-18 h after mating. Females that store few sperm are receptive to remating sooner than those storing larger number of sperm. The return of female receptivity correlates both with the decline in numbers of stored sperm as those sperm are released and used for fertilization and, in the laboratory, with longer periods of male-female pair confinement, continued availability of food, and population density (Qazi, 2003 and references therein).
Potential mechanisms for the sperm effect (depression of female receptivity) include: pressure from within the sperm storage organs exerted by sperm, movements of sperm within the storage organs, and/or distension of the uterine walls by sperm and semen. In the white cabbage butterfly Pieris rapae, receipt of sperm and seminal fluid products results in changes in female receptivity to mating. Distension of a female P. rapae uterus with saline causes a decrease in receptivity similar to that after mating. A similar mechanism in the Drosophila sperm storage organs could explain the Drosophila sperm effect. Since the sperm effect is established several hours after sperm storage is complete (10-18 h vs 1.5 h, respectively), the effect of sperm on female behaviors is proposed to involve an intermediary acting on the female central nervous system (CNS). This intermediary could be a molecule(s) secreted from the female's cells. For example, in the redbanded leafroller moth, Argyrotaenia velutinana, mating depresses female pheromone titer via a CNS-mediated release of the peptide PBAN (pheromone biosynthesis activating neuropeptide). When the female CNS is disrupted by cutting the ventral nerve cord, PBAN remains within female tissues, and is not released into the hemolymph, where it normally acts. Alternatively, the intermediary could be a molecule from the male's seminal fluid. Although most seminal proteins are not detectable in the female more than a few hours after mating, others may be stabilized by associating with sperm, allowing them to act within the female long after mating has ended (Qazi, 2003 and references therein).
In several organisms, matings are costly to females. In Drosophila, this cost arises from a combination of exposure to males, receipt of Acps, and allocation of resources to the increased egg production postmatin. Sperm themselves do not directly contribute to the cost of mating in Drosophila. However, by storing sperm, females reduce costs that accrue from repeated matings since they do not need to mate as many times to maintain fertility (Qazi, 2003 and references therein).
Despite the costs associated with multiple mating and the general reluctance of females to remate after mating, female promiscuity is well documented in Drosophila. When a female mates with more than one male, female sperm storage provides both a venue for sperm from different males to compete for access to fertilizations (sperm competition) as well as an arena for the female to select sperm from among the contributions of competing males (sperm preference or cryptic female choice). These processes may be important mechanisms preventing genetic incompatibility (the production of nonviable or infertile progeny) by selecting among male ejaculates. This is observed in Drosophila: when a D. simulans female mates with both a D. simulans and a D. mauritiana male, D. simulans sperm are preferentially stored and used for fertilization. The mechanisms of this conspecific male advantage include both the inactivation and physical displacement of D. mauritiana stored sperm (Qazi, 2003 and references therein).
Sperm competition and sperm preference have potentially important evolutionary consequences. When a female Drosophila mates with more than one male, the most recent partner usually sires more than 80% of the subsequent progeny ('last male precedence'). However, large variation exists in the fertilization success among last-mating males due to genetic differences among both males and females. Examining sperm storage sheds light on the mechanisms of sperm precedence (Qazi, 2003 and references therein).
Female sperm storage in Drosophila occurs in two types of specialized organs, located at the anterior end of the uterus. The single seminal receptacle is on the ventral side, and the paired spermathecae are on the dorsal side of the uterus. The two types of storage organs differ in morphology and in patterns of sperm storage (Qazi, 2003 and references therein).
Numerically, the seminal receptacle plays the biggest role in sperm storage, retaining 65%-80% of the stored sperm. Observations and counts of stored sperm over time indicate that sperm are released almost exclusively from the seminal receptacle for the first several days after mating. Then, as the seminal receptacle sperm become depleted, sperm are released from the spermathecae. The seminal receptacle's importance in sperm storage is supported by a phylogenetic analysis of sperm storage organ use among 113 species of Drosophila. Loss of the sperm storage function by the seminal receptacle is a rare evolutionary event that appears to have occurred only once; it characterizes only 3 (2.6%) of the species examined. In contrast, the spermathecae's sperm-storage function may have been lost as many as 13 different times affecting 38 (33.6%) of the examined species (Qazi, 2003 and references therein).
The seminal receptacle is a thin (5-20 microm) blind-ended tube that is coiled against the outer uterine wall. In Drosophila, the seminal receptacle is more than 2 mm long, slightly longer than a sperm (1.75-1.90 mm). A positive relationship also exists between sperm and seminal receptacle length among 44 other Drosophila species examined. This observation and other detailed evolutionary studies suggest that increases in seminal receptacle length drive the evolution of longer sperm (Pitnick, 1999) (Qazi, 2003 and references therein).
The lumen of the seminal receptacle is lined with a thin cuticle and is surrounded by a layer of nonsecretory cells, a basement membrane, and a helically coiled layer of muscle. There does not appear to be a sphincter separating the seminal receptacle from the uterus. At any time, only a few sperm are observed in the proximal half of the tube (that is, near its entry into the uterus). The majority of stored sperm appear partially extended and lying parallel to each other within the distal half of the seminal receptacle. This mass of sperm is curvilinear, and their tails can be seen to move, except when the seminal receptacle is very full of sperm (Qazi, 2003 and references therein).
No genes responsible for seminal receptacle length or morphology have been identified. However, results of quantitative genetic analysis of selection experiments for increased or decreased seminal receptacle length suggest that only a small number (2-5) of loci determine seminal receptacle length. Since their effect is largely additive, the loci appear to be independent (Qazi, 2003 and references therein).
Fewer sperm reside within the paired spermathecae than in the seminal receptacle (maximum of 135-449 sperm within both spermathecae). However, the paired spermathecae appear to have two important roles in sperm storage. (1) Spermathecae are the long-term storage organs. Sperm may accumulate more slowly in the spermathecae than in the seminal receptacle. As mentioned before, spermathecal sperm are apparently used for fertilization after the seminal receptacle's sperm have been depleted. (2) Spermathecae may secrete substances that maintain sperm viability within both the seminal receptacle and the spermathecae. Posterior to the spermathecae, but also opening into the dorsal side of the uterus, are two spermathecal glands whose secretions could also affect sperm storage (Qazi, 2003 and references therein).
Each spermatheca joins the uterus via a stalk composed of a thin lumen surrounded by a thick cuticular intima, epithelial cells, and a helically coiled layer of muscle cells. Like the seminal receptacle, the spermathecae do not appear to have a sphincter near their base. The spermatheca itself is an oval-shaped capsule formed from a cuticular intima. Thin ducts within the spermathecal intima open to secretory cells surrounding the exterior of the capsule. These cells produce a lamellar type of secretion that accumulates within the spermathecal lumen. Sperm move from the uterus into the spermathecal capsule through the stalk. Within the capsule, they wind around forming a toroidal mass (Qazi, 2003 and references therein).
Two genes have been identified that affect spemathecal development. Several alleles of lozenge (lz), which encodes a putative transcription factor, cause loss of the spermathecal glands and vary in the extent to which they affect spermathecal development. Some lz females have intact spermathecae, while others are missing capsules and have malformed or missing spermathecal stalks. These alleles also cause low fertility, apparently due to lower sperm storage in the seminal receptacle and the loss of motility of any stored sperm within a few days after mating. Analysis of lz mutants suggests that they impair dorsal cell migration in the early genital imaginal disc that is needed for subsequent spermathecal development. dachshund (dac), a nuclear protein, is important for appropriate formation of the spermathecal stalks. When dac expression is blocked during development, the spermathecal capsules appear normal, but they share a single stalk. In addition to these genes that affect spermathecal morphology, some genes control spermathecal number. At least two genes are responsible for the formation of extra (>2) spermathecae, but neither has yet been identified (Qazi, 2003 and references therein).
Why are there two types of sperm storage organs? Although the use of two types of storage organs is common (63.8% of 113 species examined), among Drosophila species, it is not clear why the spermathecae and seminal receptacle are both required for sperm storage. It has been suggested that the seminal receptacle might have evolved as a new organ, which is more efficient at storing sperm than are the spermathecae. The apparent coevolution of seminal receptacle length and sperm length among several Drosophila species suggests that the seminal receptacle may also provide more opportunity for female or male influence over sperm storage and sperm fate. Sperm displacement, as a result of multiple mating, appears to be primarily associated with the seminal receptacle. Residence in the spermathecae might protect sperm from displacement, and the secretions of the spermathecae might also be essential for viability (Qazi, 2003 and references therein).
When do sperm enter and exit storage? Sperm storage begins before the ~20-min copulation is complete. Sperm accumulate rapidly within the two types of storage organs, leveling off at ~700-1000 sperm less than 6 h after mating ends. The first egg laid after mating (~90 min) pushes out remaining unstored sperm. By 10 h after mating, seminal receptacle sperm numbers have noticeably declined due to the female's use of stored sperm to fertilize her eggs. On average, the number of sperm in the seminal receptacle declines at ~100-170 per day. By 48 h after mating, sperm storage in the seminal receptacle has declined by ~50%, while sperm stored in the spermathecae have decreased by only ~15% (Qazi, 2003 and references therein).
How does sperm storage occur? Sperm storage can be viewed as a series of steps: progression of sperm through the female reproductive tract after mating, entry of sperm into the storage organs, retention and maintenance of stored sperm, and release of sperm from storage up to the point of fertilization. Both female and male Drosophila play active roles in sperm storage, as shown by the nontransitivity of numbers of stored sperm 1 h after mating between different fly strains. Female-based mechanisms can include absorption of fluids from parts of her reproductive tract (called 'hydraulics' here), contractions of the uterus, contractions of the sperm storage organs, restriction of sperm to particular regions of the female reproductive tract, and/or factors secreted from within the female reproductive tract. Male-based mechanisms are also important and can continue even after copulation is complete. These include sperm motility and seminal proteins. Sperm are motile when they are transferred to the female and when they are in storage. Although it has been hypothesized that sperm motility is important for the release of sperm from storage, and it seems a likely contributor to that process, the role of sperm motility in sperm entry into storage in Drosophila is unknown. In contrast, studies have shown a profound effect on female sperm storage of secretions from the male's ejaculatory duct and accessory glands transferred to the female during mating. Matings between normal females and males transferring sperm, but no Acps, are infertile, suggesting that Acps play an essential role in sperm use once sperm are transferred to the female. When these females mate first with males transferring Acps, but no sperm, then mate with males transferring sperm, but no Acps, the second male's fertility is restored. Females mated to males that transferred sperm but greatly reduced (~1%) quantities of Acps store fewer than 10% as many sperm as do females mating to wild-type males (Qazi, 2003 and references therein).
What gets sperm into storage? Sperm may be drawn into storage by pressure changes within the female reproductive tract. In the Dipteran Culicoides melleus, and possibly in other lower Diptera, one component of sperm storage apparently involves fluid absorption from the spermathecae, which create a force that sucks sperm into storage. Although a similar mechanism could potentially function in Drosophila's seminal receptacle, ultrastructural studies argue against this hypothesis for sperm entry into the Drosophila spermathecae because substances from the surrounding cells accumulate within the spermathecae at the same time that sperm storage is occurring (Qazi, 2003 and references therein).
In many animals, female muscular contractions apparently push sperm from the uterus into storage. Consistent with this occurring in Drosophila, a Drosophila female CNS is required for sperm storage. This has been tested by manipulating expression of transformer (tra), whose product is required for normal female development. Various levels of ectopic expression of tra in either tra-deficient mutant XX flies or XY flies results in individuals possessing female genital morphology (phenotypic females), but either a masculinized CNS (evinced by male courtship behavior) or a (presumably) feminized CNS. Animals with the presumably female CNS store nearly 6.5 times more sperm within all storage organs (~550 sperm) but allocate proportionally fewer of the sperm to their seminal receptacles than do phenotypic females with a masculinized CNS. These results show that a feminized nervous system is necessary for sperm storage and that females actively distribute sperm among the storage organs. Results of experiments in which males mate with isolated female abdomens (that lack ganglia and therefore a CNS) support this hypothesis. It is possible that a female CNS is needed to trigger uterine muscle contractions that push sperm into storage. Alternatively, a female CNS could influence sperm storage by stimulating endocrine cells to release substances that attract sperm to the storage organs. Finally, the generous innervation of the seminal receptacle suggests that local contractions of the sperm storage organs might be important for sucking sperm into storage and/or for efficient arrangement of sperm within storage (Qazi, 2003 and references therein).
Sperm may be lured into storage by substances produced by the female or by male-derived substances activated once in the female. In sea urchins and other marine invertebrates, sperm-activating peptides (SAPs) secreted from the egg jelly stimulate sperm movements and orientation to eggs as well as activate other sperm-egg interactions (Suzuki 1995). Compounds within the female Drosophila reproductive tract could potentially act in a similar way, but none have been positively identified thus far (Qazi, 2003 and references therein).
Concentrating sperm to specific regions of the reproductive tract can increase the likelihood that they will encounter the openings to the storage organs. (1) Female anatomy, such as folds in the uterine wall, could channel sperm into the seminal receptacle. (2) In Drosophila females, a barrier of unknown composition exists at the base of the oviduct that keeps sperm in the uterus. At least one male seminal protein, the accessory gland protein Acp36DE, localizes at this barrier, but no chemical components required for barrier formation have been identified. In egg-less females, the barrier is mislocalized or does not form and sperm are found within the common and lateral oviducts. The presence of oocytes in the ovaries may help form the block by creating a back-pressure and/or causing the secretion of a substance that localizes near the base of the oviduct preventing sperm from premature access to oocytes within the ovary (Qazi, 2003 and references therein).
From within her reproductive tract, a female Drosophila secretes factors important for sperm storage. PubMed ID: Glucose dehydrogenase (Gld), an enzyme-producing reactive oxygen species, is secreted from the spermathecal stalks and the genital plates (located near the gonopore). gld-mutant females store fewer sperm within, and allocate sperm more unevenly between, the two spermathecae, but only when sperm storage is submaximal (<500 total sperm stored). Therefore, Gld may facilitate sperm storage, particularly when sperm are not plentiful, but is not essential for sperm storage to occur. The mechanism of Gld's sperm storage effects is unknown, but Gld is unlikely to serve as a chemoattractant since it is produced in more than one location within the female reproductive tract (Qazi, 2003 and references therein).
Corralling can also involve male contributions. The male accessory glands contain filaments composed of globular subunits. Similar looking filaments are observed in female storage organs interdigitated with stored sperm. If the filaments of similar appearance are indeed the same, those filaments might provide a scaffold along which sperm move or within which sperm are confined, to facilitate the efficient movement of sperm into storage. In an analogous way, the mating plug, contributed at least in part by the male, is thought to facilitate sperm storage by forming a physical barrier that prevents the loss of sperm from the uterus; sperm are confined above the plug, concentrating them near the entrances of the storage organs. The mating plug has also been proposed to provide a trellis to facilitate sperm movement toward storage. The mating plug contains several male seminal proteins, including PEB-me (its major component, derived from the ejaculatory bulb) and Acps, including the protein Acp36DE. Entering sperm traverse the mating plug and are limited to the anterior portion of the uterus. The mating plug is not detected more than 6 h after mating and, while its fate is unknown, it seems likely to be expelled when the first egg is laid or dissolved and reabsorbed (Qazi, 2003 and references therein).
By examining sperm storage in the presence of normal or very low amounts of Acps, it has been shown that Acps are required for sperm storage. One of these proteins, Acp36DE, is essential for proper sperm storage. Females mated to Acp36DE-deficient mutant males receive normal quantities of sperm during mating, but store far fewer sperm than females mated to wild-type males. Without Acp36DE, sperm start to enter storage at the normal time, but the subsequent accumulation of sperm into the seminal receptacle and spermathecae is less efficient. The rate at which sperm are released from spermathecal storage differs slightly in the presence or absence of Acp36DE. Within the first 24 h after mating, the rate at which sperm are released from the seminal receptacle is similar in the presence or absence of Acp36DE, but between 24-48 h after mating, proportionally more sperm are lost from the seminal receptacles of females receiving Acp36DE than females not receiving Acp36DE from their mates. It is not clear whether this phenomenon is a direct result of Acp36DE action or a secondary consequence of storing fewer sperm. The number of progeny produced in the absence of Acp36DE corresponds to the number of sperm in storage, indicating that those sperm that are stored without Acp36DE are fully viable. Thus, Acp36DE's primary role is in facilitating sperm storage. It may potentially have a secondary role in promoting sperm retention in the seminal receptacle, but there is no evidence that Acp36DE affects sperm viability (Qazi, 2003 and references therein).
Acp36DE is a novel 122-kDa glycoprotein that is transferred to females beginning within the first 5 min of mating. Within the female, it is processed to a 68-kDa product. Acp36DE is detectable within the female reproductive tract for as long as 3 h after mating. It localizes to the oviduct wall anterior to the sperm storage organ openings (at the barrier discussed above) and on the anterior end of the mating plug. Thus, it is present at the upper and lower areas of the 'corral' described above. In addition, Acp36DE enters the sperm storage organs. Finally, Acp36DE binds to sperm in vivo and in vitro. When the first egg is laid, the Acp36DE in the oviduct and mating plug are expelled. Although no longer detectable within the female, Acp36DE donated from one male facilitates the storage of a second male's sperm 24-48 h later (Qazi, 2003 and references therein).
The available data on Acp36DE support several, not mutually exclusive, models for its action. Acp36DE may interact with targets on female tissues stimulating females to push (from the uterus) or suck (from the sperm storage organs) sperm into storage via muscular contractions. It could potentially also stimulate the release of chemoattractant molecules. Alternatively, Acp36DE may limit sperm movements via its associations with the oviduct, mating plug, and sperm, thereby facilitating the rapid accumulation of sperm within storage. Finally, Acp36DE might help track sperm into storage by forming a scaffold or providing guidance cues along which the sperm move, or are moved, into storage (Qazi, 2003 and references therein).
What keeps sperm viable in storage? Once Drosophila sperm are in storage, they need to remain viable for up to 2 weeks. For example, even in cases of matings between genetically incompatible strains of Drosophila, sperm in the storage organs remain viable several days after mating (suggested by vital cell staining). Both female and male factors probably contribute to the maintainance of sperm in storage. Substances produced within the spermathecae may play roles in sperm viability and retention. The presence of the spermathecal capsule correlates with female fertility. The low and variable fertility of some female lz mutants suggests a similar model for Drosophila, since the lz mutant phenotype is proposed to be due to the presence/absence of the spermathecal capsule. Females with at least 1 spermathecal capsule produced an average of 216 progeny, 36 times as many progeny as mutant females lacking spermathecal capsules. Lower fertility is attributable to a shorter duration of progeny production among females lacking capsules compared with their normal sisters. Although the seminal receptacle appears normal, lz mutants store fewer sperm which lose motility earlier (less-than or equal to 5 days after mating) than do sperm in wild-type females (>11 days). These results suggest that presence of the secretory cells surrounding the spermathecal capsule and/or the spermathecal glands is important for the viability of stored sperm and for female fertility (Qazi, 2003 and references therein).
Stored sperm need to be protected from degradation. During storage, proteolysis of sperm surface proteins could destroy a sperm's ability to bind to eggs; alternatively, regulated proteolysis of the surface of stored sperm could be essential to activate or capacitate them. Indeed, in mice, mutations in seminal fluid protease inhibitors impair fertility, consistent with the hypothesis that protease inhibitors serve to protect sperm. Drosophila seminal fluid also contains regulators of proteolysis. Of ~83 predicted secreted male accessory gland proteins, 9 are predicted (or demonstrated) regulators of proteolysis. One, the trypsin inhibitor Acp62F, has been shown to enter the mated female's sperm storage organs, consistent with its playing a role in protecting sperm from degradation. Stored sperm are also potentially subject to untoward effects from microbes that might have entered the female's genital tract during mating. Perhaps to guard against this, sperm storage organs and seminal fluids contain antimicrobial peptides. The seminal receptacle and spermathecae also both secrete Drosomycin, a peptide with antifungal properties (Qazi, 2003 and references therein).
What releases sperm from storage? Sperm leave storage to two potential fates: one fate is to fertilize an ovulated egg, another is to leave storage but not to fertilize an egg either due to inefficient sperm use or to sperm displacement as a result of the female mating with another male (female sperm preference or sperm competition). Since sperm use in Drosophila is efficient, one (or very few) sperm leave(s) storage to fertilize an ovulated egg as the egg comes to rest in the uterus. The egg lodges there with its anterior end, containing its micropyle, close to the sperm storage organ entrances. It is not known whether sperm swim, are pushed, or are sucked through the micropyle. The entire sperm enters the egg. Its tail coils in the anterior end and persists within the embryo until shortly after hatching (Qazi, 2003 and references therein).
In Drosophila, conformational changes of the reproductive tract induced by ovulation might also effect sperm release. Single sperm have been observed near the opening of the seminal receptacle as an egg passes down the oviduct; it has been proposed that ovulation and sperm release are correlated. Muscular contractions of just the seminal receptacle could squeeze small numbers of sperm out of storage (Qazi, 2003 and references therein).
Motile sperm might motor their way out of storage. Sperm circulating within the storage organs have been observed and it has been speculated that, occasionally, a single sperm leaving storage would encounter an egg. This observation, coupled with the lack of detected sphincters at the base of the sperm storage organs, has led to the proposition that sperm motility aids their release (Qazi, 2003 and references therein).
At least one male-derived protein is suggested to play a role in sperm residence in storage in females: the carboxylesterase, Esterase-6 (Est-6). Est-6 is secreted from the male ejaculatory bulb and anterior ejaculatory duct, and is transferred to the female early during mating. Activity of male-derived Est-6 is detected for only 2 h after mating. Although the initial timing and storage of sperm into females that do or do not receive Est-6 from their mates is similar over time, more sperm are retained in females that do not receive Est-6. Est-6 therefore appears to play a role in the release of sperm from storage in the seminal receptacle; its role on spermathecal sperm is unclear. Est-6 might cause the release of sperm from the seminal receptacle by affecting sperm motility within the sperm storage organs or by catalyzing the production of molecules needed to sustain sperm motility. However, since Est-6 activity is also positively correlated with the rate of female oviposition as well as female latency to remating, and since Est-6 enters the female hemolymph, it may have more than one target or multiple interrelated effects (Qazi, 2003 and references therein).
If a female mates twice, the second male's ejaculate causes the release of previously stored sperm (last male sperm precedence). This effect is attributable to the removal of some sperm from storage and the inactivation of remaining sperm. The method of displacement depends on the time between matings. If a female remates within 2 days of an initial mating, the last-mating male's sperm plays a role in physical displacement of sperm, primarily from the seminal receptacle. With longer intervals between rematings, other seminal components, particularly Acps from the most recently mating male, functionally displace sperm by decreasing the use of previously stored sperm that remain after the second mating. If seminal proteins temporarily protected the male's sperm from displacement, then perhaps it is the inactivation of these 'protein companions' to sperm that leave the sperm vulnerable to displacement by future mating males. Males lacking Acp36DE are poor displacers of other males' sperm and often have their own sperm nearly completely displaced from storage (Chapman, 2000), but this is believed to be because they are poor at getting their own sperm moved into storage (Qazi, 2003 and references therein).
Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. This paper reports identification of the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC): Spermathecal endopeptidase 1 (Send1), which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2), which is induced by mating. These regulatory sequences were used to perform precise genetic ablations of the SSC at distinct time points relative to mating. The SSC were shown to be required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1) reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2) causes ovoviviparity -- the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects (Schnakenberg, 2011).
Females of many animal species store sperm after mating, in specialized organs of the reproductive tract. In addition to sperm, seminal proteins are transferred to females during mating. In Drosophila, seminal proteins perform multiple functions that advance the male's reproductive interests. These functions include promoting sperm storage, decreasing the female's receptivity to subsequent courters, and stimulating egg production and ovulation (Schnakenberg, 2011).
Female reproductive interests do not necessarily coincide with those of their mates. A coevolutionary arms race can therefore ensue. In Drosophila, the coevolutionary pressure on female reproductive functions is apparently quite strong: seminal fluid decreases female lifespan and does so to a greater extent when females are experimentally prevented from coevolving with males. Of course, males and females do share some reproductive interests, such as successful production of offspring, and therefore evolutionary pressure also exists to coordinate their reproductive functions. Although it has been appreciated that molecular interactions -- both antagonistic and cooperative -- between male and female products are key to understanding insect fertility and its evolution, much more progress has been made in characterizing the composition and functions of seminal fluid than in characterizing female reproductive secretions (Schnakenberg, 2011).
A potentially major role of female secretions is in sperm storage. Insect females typically have multiple specialized sperm storage organs, to which sperm are recruited after copulation and in which sperm can be maintained for weeks or, in the case of queens of social insect species, years. Female Drosophila melanogaster have three such organs located at the anterior of the uterus: a long tubular seminal receptacle and a pair of spermathecae. Each spermatheca is mushroom shaped, with a duct that extends from the uterus to a cuticular cap. The seminal receptacle houses up to 80% of stored sperm, whereas the spermathecal caps house the remainder. Each spermathecal cap is lined with large glandular cells containing prominent secretory organelles that open into the lumen where sperm are stored. Despite considerable divergence in sperm-storage organ anatomy, such cells are found lining or adjacent to the spermathecae of a wide range of insects. The position of these spermathecal secretory cells (SSC) suggested they might have a role in sperm storage, yet direct in vivo evidence has been lacking. Indeed, the most direct evidence for a role in sperm storage comes from a 1975 study of boll weevils with surgically removed spermathecal glands: sperm did not enter the spermatheca in such females, although because sperm motility was greatly diminished it cannot be concluded whether the glands are necessary just for sperm viability or also for recruitment into storage (Schnakenberg, 2011).
The SSC might also contribute to sustained levels of egg production and fertilization, by secreting proteins that alter female reproductive physiology or by modulating the activities of male seminal proteins. Sex peptide, a seminal protein, binds to sperm tails and during storage is gradually cleaved to an active form that stimulates egg production. Sex peptide is also required for release of sperm from storage. In the female, the sex peptide receptor is expressed in neurons that mediate a decrease in courtship receptivity and an increase in egg laying after mating, and it is required for these changes. The sex peptide receptor is also expressed in the SSC, suggesting that sex peptide acts directly on these cells (Schnakenberg, 2011).
Other genes expressed specifically in the SSC have not been comprehensively identified, although some such genes have emerged from transcriptional profiling studies of: (1) somatic tissues of males versus females, (2) dissected whole spermathecae, and (3) virgin versus mated females. One notable class of genes revealed by these studies is those encoding proteases. Protease-encoding genes are over-represented among those induced in females by mating, and among those highly expressed in the spermathecae. Proteases are especially interesting due to their potential for interactions with seminal proteins. Such interactions could be antagonistic, for example by degradation of seminal proteins, or cooperative, for example by regulated cleavage of seminal proteins to their active forms. Male-female coevolution would be expected to lead to rapid divergence of female-expressed protease-encoding genes, and indeed such genes show elevated rates of coding-sequence evolution in several species (Schnakenberg, 2011).
To address how the SSC contribute to female postmating physiology, tools have been developed that allow manipulation of these cells in a precise spatiotemporal manner. To develop these tools, the regulatory regions were identified controlling transcription of two protease-encoding genes that are expressed exclusively in the SSC. The gene CG17012, which encodes a serine-type endopeptidase, is expressed exclusively in the SSC, in both unmated and mated females. CG17012 is refered as Spermathecal endopeptidase 1 (Send1). CG18125, which also encodes a serine-type endopeptidase, is expressed exclusively in the SSC and its transcription is upregulated 76-fold 3-6 h after mating. CG18125 is refered as Spermathecal endopeptidase 2 (Send2). Identifying these genes' regulatory regions allowed creation of drivers and reporters for manipulating and monitoring the SSC (Schnakenberg, 2011).
This study shows that the SSC are required to recruit sperm to the spermathecae, but not for maintaining them there. Moreover, the SSC act at a distance in the reproductive tract, in that they are required for maintaining sperm motility in the seminal receptacle. Action at a distance was also shown with respect to egg laying, in that females lacking SSC are ovoviviparous. Fertilized eggs develop, and indeed sometimes hatch into larvae, inside the uterus. This phenotype is reminiscent of two species of Drosophila that retain developing eggs, D. sechellia and D. yakuba. These results therefore not only reveal the functions of a poorly understood reproductive tissue, but also shed light on the evolution of live birth (Schnakenberg, 2011).
For each of the SSC-expressed genes, Send1 and Send2, a driver was created carrying ~4 kb of upstream sequence and ~4 kb of downstream sequence, flanking the coding sequence of the yeast transcriptional activator GAL4, which can activate transgene expression in Drosophila through its cognate UAS sequence. GAL4-independent reporters were also created by cloning a subfragment of the Send1 or Send2 upstream sequence in front of the coding sequence of a fast-maturing, nuclear-localized, red-fluorescent protein (Schnakenberg, 2011).
Send1-GAL4 drives expression of a membrane-bound green fluorescent protein (GFP) (UAS-mCD8-GFP) specifically in the SSC of virgin and mated females. GFP expression driven by Send1-GAL4 is visible by 20 h posteclosion and increases in intensity by day 4. Send2-GAL4 drives GFP expression in the SSC of mated females only, as early as 3 h postmating. Send1-nRFP and Send2-nRFP recapitulate expression of the respective endogenous genes as well (Schnakenberg, 2011).
The potential for redundancy among spermathecae-expressed serine proteases is high. Protease-encoding genes are over-represented among those induced in females by mating, and among those highly expressed in the spermathecae. Moreover, some spermathecae-expressed serine proteases are recently duplicated paralogs with high levels of amino acid identity. Consistent with redundancy, no effects were observed on female fecundity or fertility when Send1-GAL4 was used to drive RNAi efficiently targeting Send1 or Send2 transcripts (Schnakenberg, 2011).
To fully understand how SSC-expressed proteases contribute to female reproductive function might require the simultaneous knockdown or knockout of many genes. As an alternative approach, the Send1-GAL4 and Send2-GAL4 drivers were used to ablate the SSC at different times, thereafter eliminating their ability to secrete any products into the spermathecal lumen. As the SSC are terminally differentiated adult cells, the drivers were used to express a modified form of the apoptosis-promoting protein Hid (HidAla5) that is effective in postmitotic cells (Schnakenberg, 2011).
Sperm storage was examined in SSC-ablated and control females by individually mating them with males expressing protamine-GFP, which renders sperm heads fluorescent green. Males transfer between 3,000 and 4,000 sperm during mating, of which only ~25% are stored. Of the stored sperm, approximately 65% to 80% reside in the seminal receptacle and the rest reside in the spermathecae. Mortality of stored sperm remains quite low for about 2 wk. In control +/UAS-hidAla5; Sp/+; Send1-nRFP/+ females, sperm are stored in the spermathecae and seminal receptacle within 1 h of mating, although sperm are also found in the uterus and occasionally the oviduct. In their +/UAS-hidAla5; CyO, Send1-GAL4/+; Send1-nRFP/+ sisters, whose SSC were ablated prior to mating, sperm are also found in the seminal receptacle and uterus, and occasionally in the oviduct, but often are not present in the spermathecae. Indeed, out of 17 SSC-ablated females, 16 had at least one empty spermatheca, and eight of these had both spermathecae empty. By contrast, no control female had even one empty spermatheca, and in only one case did one of the spermathecae contain fewer than ten sperm. In cases of SSC-ablated females in which sperm were found in one of the spermathecae but not the other, the presence or absence of sperm correlated with the presence or absence of SSC in a mosaic female. These results imply that the SSC are required to recruit sperm to the spermathecae (Schnakenberg, 2011).
It was previously shown that glucose dehydrogenase, which is secreted from the proximal and distal ends of the spermathecal duct, promotes recruitment of sperm into the spermathecae. Some cases were observed in which sperm were present in the spermathecal duct leading to a spermathecal cap with no sperm, but in most cases the ducts were empty of sperm as well. This result suggests that recruitment of sperm by the duct cells is largely dependent on SSC function (Schnakenberg, 2011).
By 7 h postmating, nearly all remaining sperm in control females are in the seminal receptacle or spermathecae, as expected. By contrast, SSC-ablated females have sperm in their seminal receptacles, yet tend to lack sperm in the spermathecae. The lack of sperm in the spermathecae at 7 h suggests that sperm recruitment to the spermathecae is indeed impaired and not merely delayed (Schnakenberg, 2011).
At 24 h postmating, sperm storage in control females appears very similar to that observed at 7 h postmating. In SSC-ablated females, however, sperm dynamics in the seminal receptacle are aberrant. Whereas in control females, sperm are found throughout the tubular receptacle, in many SSC-ablated females, sperm have lost motility and clumped together in one part of the receptacle, leaving the rest of it largely empty. This result suggests that the products of the SSC travel to, and act in, the seminal receptacle. Consistent with this inference, females lacking entirely the spermathecae and female accessory glands lose fertility within a few days after mating. The loss of fertility is apparently caused by loss of motility of sperm stored in the females' seminal receptacles. These results localize the source of at least one motility-maintaining factor to the SSC (Schnakenberg, 2011).
At 6 to 9 d postmating, sperm storage in control females still appears very similar to that observed at 7 h or 24 h postmating. Likewise, sperm storage at 6 to 9 d postmating in SSC-ablated females appears very similar to that observed in SSC-ablated females at 24 h postmating. Although rare females contain a few sperm in their spermathecae, most do not . As at 24 h postmating, clumps of sperm in the seminal receptacle are also seen in some females (Schnakenberg, 2011).
The existence of a few sperm in the spermathecae of some SSC-ablated females approximately 1 wk postmating suggests that the SSC are not required to retain sperm in the spermathecae once they have been stored there. However, as described above, there is a correlation between sperm recruitment to the spermathecae and the existence of residual, nonablated SSC. It could be that the same residual SSC function that recruited the sperm to the spermathecae is sufficient to retain them there. To test definitively whether the SSC are required to retain sperm in the spermathecae, the SSC were eliminated after sperm were stored in the spermathecae. This was done by ablating the SSC after mating using Send2-GAL4 in combination with UAS-hidAla5 (Schnakenberg, 2011).
At 7 h postmating, sperm storage in control +/UAS-hidAla5; Send1-nRFP/+; MKRS/+ females does not appear different to that of their +/UAS-hidAla5; Send1-nRFP/+; Send2-GAL4/+ sisters. As noted above, SSC ablation is complete in the latter females within one more day. If the SSC are required for long-term sperm retention in the spermathecae, then SSC-ablated females should lose the sperm they had stored in the spermathecae. However, this is not the case. At 6-8 d postmating, SSC-ablated females retain as many sperm in their spermathecae as do their control sisters. Moreover, sperm clumping in the seminal receptacle is not observed in these SSC-ablated females, implying that whatever SSC products are required to maintain sperm motility need only be supplied up to or around the time of mating, not continually (Schnakenberg, 2011).
Because females whose SSC are ablated prior to mating do not store sperm in their spermathecae and lose sperm motility in their seminal receptacles, it was next asked whether this impaired sperm storage affects fecundity or fertility. Females with SSC ablated prior to mating lay as many eggs on days 1 to 3 postmating as their control sisters. However, after day 3, their egg laying is significantly reduced. Notably, after day 3 an individual SSC-ablated female tends to lay vastly different numbers of eggs on successive days. Indeed, 10 out of 18 SSC-ablated females had one day in which 0 or 1 egg was laid, followed immediately by a day with greater than ten eggs laid. By contrast, only one out of 15 control females had any day in which 0 or 1 was laid (Schnakenberg, 2011).
The alternation of low and normal levels of egg laying in SSC-ablated females suggests that the SSC play some role in either ovulation or oviposition. To determine which is the case, SSC-ablated females that had not laid an egg in the previous 24 h were dissected. Strikingly, SSC-ablated females are ovoviviparous: a large proportion of such females (eight out of 16) had a late-stage embryo or live first-instar larva stuck in the uterus. This result implies a defect in ejecting eggs from the uterus (oviposition) rather than egg production and release (ovulation). However, those females with stuck eggs did not appear to have a 'log jam' of eggs in the oviduct, suggesting either: (1) that inability to eject a fertilized egg signals back to the ovary to halt or slow ovulation; or (2) that ovulation is independently slowed by SSC loss (Schnakenberg, 2011).
A simple, mechanical explanation for the stuck-egg phenotype is that the SSC produce a lubricant that coats the uterus, allowing eggs to pass easily. An alternative explanation is that products of the SSC are required before or around the time of mating to trigger cellular and physiological changes in the reproductive tract that are required for full reproductive maturity. Consistent with the latter explanation, females whose SSC have been ablated postmating, using Send2-Gal4, do not show any difference from control sisters in the number of eggs laid on each of days 1 to 8 postmating. This result implies that proper egg laying after day 3 postmating requires SSC function earlier in adulthood. The earlier function could be the production of a secretion, such as a lubricant, that is long-lived. However, multiple lines of evidence support the existence of posteclosion and postmating developmental programs by which the female reproductive tract achieves full maturity. The triggering of such a program by one or more SSC gene products could explain not only the egg-laying defect but also the loss of sperm motility in the seminal receptacle in females with Send1-driven, but not Send2-driven, ablation of the SSC (Schnakenberg, 2011).
Work prior to this had suggested several possible functions for the secretory cells of the spermathecae, including recruitment and maintenance of sperm, yet direct in vivo evidence was lacking because of the absence of tools for precisely manipulating these cells. In D. melanogaster, it had been observed that females lacking the spermathecae and accessory glands lose fertility, despite having normal seminal receptacles, but this effect could not be ascribed to any particular cell population within the missing organs. These cell-specific drivers enabled determination that the SSC contribute to reproductive function in multiple ways, some expected and some not. The SSC do indeed produce one or more products required for recruiting sperm into storage, although they are not required to maintain sperm in the spermathecae once recruited. In contrast, the SSC are not required for sperm to reach the seminal receptacle, but they are required to maintain sperm motility there, consistent with the lost fertility of females lacking spermathecae and accessory glands. In addition to their action at a distance in the seminal receptacle, the SSC also act at a distance in sustaining egg laying. The impairment of egg laying in SSC-ablated females manifests as an unanticipated phenotype: ovoviviparity. SSC-ablated females retain fertilized eggs, which develop inside the uterus and, in some cases, hatch as larvae inside the mother (Schnakenberg, 2011).
These findings have relevance to two evolutionary patterns observed in the genus Drosophila. First, all Drosophila species that have been examined have a pair of spermathecae and a single seminal receptacle, yet there are at least 13 independent Drosophila lineages in which females use only the seminal receptacle to store sperm. In species that do not store sperm in the spermathecae, the spermathecal caps are small and weakly sclerotized, but are surrounded by large cells that are presumably their SSC. The finding that the SSC act at a distance in the female reproductive tract might explain why these species retain their spermathecae, despite not using them to store sperm (Schnakenberg, 2011).
Second, the ovoviviparity observed in SSC-ablated females suggests that transitions to live birth might require fewer evolutionary steps than once thought. In a surprising recent discovery it was found that two species of Drosophila are ovoviviparous: even when exposed to ample substrate for oviposition, females of D. sechellia and D. yakuba retain fertilized eggs, which develop internally, in contrast to those of all other examined Drosophila species, which are laid immediately after fertilization (Markow, 2009). SSC-ablation results suggest that the Drosophila uterus is 'preadapted' to support internal embryo development, in that eggs stuck there are capable of hatching into perfectly viable larvae (Schnakenberg, 2011).
The genetic tools that this study has developed will be useful for further dissection of the molecular and evolutionary mechanisms underlying female reproductive function. Insect reproductive secretions are of particular interest because of their relevance to the fertility of agricultural pests and human disease vectors. To date, attention has focused on male secretions, because of the far greater knowledge of male seminal proteins than of products of the female reproductive tract, but recent studies in malaria-vector mosquitoes have begun to counter this bias. Reproductive secretions are also highly relevant to the study of beneficial insects, as evidenced by recent work characterizing the seminal-fluid and spermathecal-fluid proteomes of honey bees. Increased knowledge of the regulation and functions of spermathecal secretions will add a new dimension both to insect-control efforts and to the maintenance of healthy breeding populations of agriculturally important insects (Schnakenberg, 2011).
Sperm storage plays a key role in the reproductive success of many sexually-reproducing organisms, and the capacity of long-term sperm storage varies across species. To date there are no empirical tests of the reproductive consequences of additional long-term sperm storage. While Dipterans ancestrally have three long-term sperm organs, known as the spermathecae, Drosophila contain only two. This study identified a candidate gene, spermathreecae (sp3), in which a disruption cause the development of three functional spermathecae rather than the usual two in Drosophila. This disruption was used to test the reproductive consequences of having an additional long-term sperm storage organ. Compared to females with two spermathecae, females with three spermathecae store a greater total number of sperm and can produce offspring a greater length of time. However, they did not produce a greater total number of offspring. Thus, additional long-term sperm storage in insects may increase female fitness through extending the range of conditions where she produces offspring, or through increasing the quality of offspring via enhanced local sperm competition at fertilization (Dhillon, 2020).
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).
Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. This study used mass spectrometry to identify the single cleavage site in Acp36DE. This site was then mutated and the effects on sperm storage were tested. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin (Avila, 2017).
Spermathecae are glandular organs in
insect female reproductive tract and play essential roles for insect
reproduction; however, the molecular mechanism involved in their
development is largely unknown. Drosophila spermathecae consist
of class-III secretory units, in which each secretory cell discharges its
products to the central lumen through an end-apparatus and a canal.
Secretory unit formation in Drosophila spermathecae utilizes a
fixed cell lineage, in which each secretory unit precursor (SUP) divides
to produce one pIIb cell and one pIIa cell. The former differentiates into
an apical cell (AC), whereas the latter divides again to produce a
secretory cell (SC) and a basal cell (BC). It is unclear how each cell
acquires its identity and contributes to secretory unit formation. This
study demonstrates that Notch signaling
is required and sufficient for the specification of lumen epithelial
precursors (LEPs; vs. SUPs), pIIb (vs. pIIa), and SCs (vs. BCs)
sequentially. Notch activation in LEPs and SCs apparently utilizes
different ligand mechanism. In addition, Notch signaling both suppresses
and activates transcription factors Hindsight
(Hnt) and Cut during spermathecal lineage
specification, supporting the notion that Notch signaling can have
opposite biological outcomes in different cellular environment.
Furthermore, LEP-derived epithelial cells (ECs) and ACs show distinct
cellular morphology and are essential for securing secretory units to the
epithelial lumen. These data demonstrate for the first time the dynamic
role of Notch signaling in binary cell fate determination in Drosophila
spermathecae and the role of ECs and ACs in secretory unit formation (Shen, 2017). In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. Previous work has identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. This study used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. Normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization (Grewal, 2021).
Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction
Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. This study demonstrates that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torso(Deg)) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torso(Deg) in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction (Colonnetta, 2021).
Following fertilization, a Drosophila embryo undergoes 14 consecutive nuclear divisions to give rise to the cellular blastoderm. While the initial nuclear divisions take place in the center of the embryo, the nuclei begin to migrate toward the periphery around nuclear cycle (NC) 4-6 and reach the cortex at NC9/10. Even before bulk nuclear migration commences, a few nuclei move toward the posterior of the embryo, enter a specialized, maternally derived cytoplasm known as the pole plasm, and induce the formation of pole buds (PBs). The centrosomes associated with these nuclei trigger the release of pole plasm constituents from the posterior cortex and orchestrate precocious cellularization to form the primordial germ cells (PGCs), the progenitors of the germline stem cells in adult gonads. Unlike pole cell nuclei, somatic nuclei continue synchronous divisions after they reach the surface of the embryo until NC 14 when they cellularize (Colonnetta, 2021).
The timing of cellularization is not the only difference between the soma and PGCs. Although newly formed PGCs divide after they are formed, they undergo only one or two asynchronous divisions before exiting the cell cycle. Another key difference is in transcriptional activity. Transcription commences in the embryo during NC 6-7 when a select number of genes are active. Transcription is more globally upregulated when the nuclei reach the surface, and by the end of NC 14, zygotic genome activation (ZGA) is complete. This transition is marked by high levels of phosphorylation of residues Serine 5 (Ser5) and Serine 2 (Ser2) in the C-terminal domain (CTD) of RNA polymerase II. By contrast, in newly formed PGCs, transcription is switched off, and PGC nuclei have only residual amounts of Ser5 and Ser2 CTD phosphorylation. Moreover, and consistent with their transcriptionally quiescent status, other changes in chromatin architecture that accompany ZGA are also blocked in PGCs (Colonnetta, 2021).
Three different genes, nanos (nos), polar granule component (pgc), and germ cell-less (gcl), are known to be required for establishing transcriptional quiescence in newly formed PGCs. The PGCs in embryos derived from mothers carrying mutations in these genes fail to inhibit transcription, and this compromises germ cell specification and disrupts germ cell migration. (As these are maternal effect genes, embryos derived from nos/pgc/gcl mothers display the resulting mutant phenotypes and will be referred to as nos/pgc/gcl here onwards.) Interestingly, these three genes share only a few targets, suggesting overlapping yet distinct mechanisms of action. Nos is a translation factor and thus must block transcription indirectly. Together with the RNA-binding protein Pumilio (Pum), Nos interacts with recognition sequences in the 3'-untranslated regions (3'UTRs) of mRNAs and inhibits their translation. Currently, the key mRNA target(s) that Nos-Pum repress to block transcription is unknown; however, in nos and pum mutants, PGC nuclei display high levels of Ser5 and Ser2 CTD phosphorylation and activate transcription of gap and pair-rule patterning genes and the sex determination gene Sex-lethal (Sxl). pgc encodes a nuclear protein that binds to the transcriptional elongation kinase p-TEFb, blocking Ser5 CTD phosphorylation. In pgc mutant pole cells, Ser5 phosphorylation is enhanced, as is transcription of several somatic genes, including genes involved in terminal patterning (Colonnetta, 2021).
While the primary function of nos and pgc appears to be blocking ZGA in PGCs, gcl has an earlier function, which is to turn off transcription of genes activated in somatic nuclei prior to nuclear migration. Targets of gcl include two X-chromosome counting elements (XCEs), scute (sc/sis-b) and sisterless-a (sis-a), that function to turn on the sex determination gene, Sxl, in female soma. gcl embryos not only fail to shut off sis-a and sis-b transcription in PBs, but also show disrupted PGC formation. In some gcl embryos, PGC formation fails completely, while in other embryos only a few PGCs are formed. In this respect, gcl differs from nos and pgc, which have no effect on the process of PGC formation, but instead interfere with the specification of PGC identity (Colonnetta, 2021).
Studies by Leatherman (2002) suggested that the defects in PGC formation in gcl mutant embryos are linked to failing to inhibit somatic transcription. That study found that when PBs first form during NC 9 in wild-type (WT) embryos, levels of CTD phosphorylation PB are only marginally less than in nuclei elsewhere in the embryo. However, by NC 10, there was a dramatic reduction in CTD phosphorylation even before PBs cellularize. By contrast, in gcl mutant embryos, about 90% of the NC 10 PB nuclei had CTD phosphorylation levels approaching that of somatic nuclei. Moreover, this number showed an inverse correlation with the number of PGCs in blastoderm stage gcl embryos. Whereas WT blastoderm embryos have >20 PGCs per embryo, gcl embryos had on average just three PGCs under their culturing conditions. Interestingly, expression of the mouse homologue of Gcl protein, mGcl-1, can rescue the gcl phenotype in Drosophila (Leatherman, 2000). Supporting the conserved nature of the involvement of Gcl during transcriptional suppression, a protein complex between mGcl-1 and the inner nuclear membrane protein LAP2β is thought to sequester E2F:D1 to reduce transcriptional activity of E2F:D1 (Colonnetta, 2021).
The connection Leatherman postulated between failing to turn off ongoing transcription and defects in PGC formation in gcl mutants is controversial and unresolved. This model predicts that a non-specific inhibition of polymerase II should be sufficient to rescue PGC formation in gcl embryos. However, the PGC formation defects seen in gcl embryos are not rescued after injection of the RNA polymerase inhibitor, α-amanitin. Since α-amanitin treatment disrupted somatic cellularization without impacting PGC formation in WT embryos, it was concluded that it effectively blocked polymerase transcription. On the other hand, subsequent experiments by Pae (2017) raised the possibility that inhibiting transcription in pole cell nuclei is a critical step in PGC formation. The Pae paper showed that Gcl is a substrate-specific adaptor for a Cullin3-RING ubiquitin ligase that targets the terminal pathway receptor tyrosine kinase, Torso, for degradation. The degradation of Torso would be expected to prevent activation of the terminal signaling cascade in PGCs. In the soma, Torso-dependent signaling activates the transcription of several patterning genes, including tailless, that are important for forming terminal structures at the anterior and posterior of the embryo. Thus, by targeting Torso for degradation, Gcl would prevent the transcriptional activation of terminal pathway genes by the MAPK/ERK kinase cascade in PGCs. Consistent with this possibility, simultaneous removal of gcl and either the Torso ligand modifier, torso-like (tsl) or torso resulted in rescue of germ cell loss induced by gcl. Surprisingly, however, Pae (2017) was unable to observe a similar rescue of gcl phenotype when they used RNAi knockdown to compromise components of the MAP kinase cascade known to act downstream of the Torso receptor. Based on these findings, they proposed that activated Torso must inhibit PGC formation via a distinct non-canonical mechanism that is both independent of the standard signal transduction pathway and does not involve transcriptional activation (Colonnetta, 2021).
The current study has revisited these conflicting claims by examining the role of Gcl in establishment/maintenance of transcriptional quiescence. The studies of Leatherman (2002) indicated that two of the key X chromosomal counting elements, sis-a and sis-b, were inappropriately expressed in gcl PBs and PGCs. Since transcription factors encoded by these two genes function to activate the Sxl establishment promoter, Sxl-Pe, in somatic nuclei of female embryos, their findings raised the possibility that Sxl might be ectopically expressed in PBs/PGCs of gcl embryos. This study shows that in gcl embryos, Sxl transcription is indeed inappropriately activated in PBs and newly formed PGCs. Moreover, ectopic expression of Sxl in early embryos disrupts PGC formation similar to gcl. Supporting the conclusion that Sxl is a biologically relevant transcriptional target of Gcl, PGC formation defects in gcl embryos can be suppressed either by knocking down Sxl expression using RNAi or by loss-of-function mutations. As reported by Pae (2017), this study found that loss of torso-like (tsl) in gcl embryos suppresses PGC formation defects. However, consistent with a mechanism that is tied to transcriptional misregulation, rescue is accompanied by the reestablishment of transcriptional silencing in gcl PGCs. Lending further credence to the idea that transcription misregulation plays an important role in disrupting PGC development in gcl embryos, this study found that expression of a mutant form of Torso that is resistant to Gcl-dependent degradation (hereafter referred to as torsoDeg: Pae, 2017) ectopically activates transcription of two Gcl targets, sis-b and Sxl, in PB and PGC nuclei. In addition, stabilization of Torso in early PGCs also mimics another gcl phenotype, the failure to properly sequester key PGC determinants in PBs and newly formed PGCs (Colonnetta, 2021).
gcl differs from other known maternally deposited germline determinants in that it is required for the formation of PBs and PGCs. gcl PGCs exhibit a variety of defects during the earliest steps in PGC development. Unlike WT, gcl PGCs fail to properly establish transcriptional quiescence. While other genes like nos and pgc are required to keep transcription shut down in PGCs, their functions only come into play after PGC cellularization. By contrast, gcl acts at an earlier stage beginning shortly after nuclei first migrate into the posterior pole plasm and initiate PB formation. In gcl PBs, ongoing transcription of genes that are active beginning around nuclear cycle 5-6 is not properly turned off. This is not the only defect in germline formation and specification. As in WT, the incoming nuclei (and the centrosomes associated with the nuclei) trigger the release of the pole plasm from the posterior cortex. However, instead of sequestering the germline determinants in PBs so that they are incorporated into PGCs during cellularization, the determinants disperse into the soma where they become associated with the cytoplasmic territories of nearby somatic nuclei. There are also defects in bud formation and cellularization. Like the release and sequestration of germline determinants, these defects have been linked to the actin cytoskeleton and centrosomes (Colonnetta, 2021).
Two models have been proposed to account for the PGC defects in gcl mutants. In the first, Leatherman (2002) attributed the disruptions in PGC development to a failure to turn off ongoing transcription. The second argues that the role of gcl in imposing transcriptional quiescence is irrelevant. Instead, the defects are proposed to arise from a failure to degrade the Torso receptor. In the absence of Gcl-dependent proteolysis, high local concentrations of the Tsl ligand modifier at the posterior pole would activate the Torso receptor. According to this model, the ligand-receptor interaction would then trigger a novel, transcription-independent signal transduction pathway in PBs and PGCs that disrupts their development. These conflicting models raise several questions. Does gcl actually have a role in establishing transcriptional quiescence in PBs and PGCs? If so, is this activity relevant for PB and PGC formation? Is the stabilization of Torso in gcl mutants responsible for the failure to shut down transcription in PBs and PGCs? If not, does gcl target a novel, transcription-independent but Torso-dependent signaling pathway? Is the stabilization of Torso responsible for some of the other phenotypes that are observed in gcl mutants? These studies have addressed these outstanding questions, leading to a resolved model of Gcl activity and function (Colonnetta, 2021).
Shutting off transcription is, in fact, a critical function of Gcl protein. As previously documented by Leatherman, this study found that several of the key X-linked transcriptional activators of Sxl-Pe are not repressed in newly formed PBs and early PGC nuclei, and Sxl-Pe transcription is inappropriately activated in the presumptive germline. Previous studies found that ectopic expression of Sxl in nos mutants disrupts PGC specification. In this case, the specification defects in nos embryos can be partially rescued by eliminating Sxl activity. The same is true for gcl mutants: elimination or reduction in Sxl function ameliorates the gcl defects in PGC formation/specification. Conversely ectopic expression of Sxl early in embryogenesis mimics the effects of gcl loss on PGC formation. Importantly, the role of Gcl in inhibiting Sxl-Pe transcription is not dependent upon other constituents of the pole plasm. When Gcl is ectopically expressed at the anterior of the embryo, it can repress Sxl. This observation is consistent with the effects of ectopic Gcl on the transcription of other genes reported by Leatherman et al., 2002. Since the rescue of gcl by eliminating the Sxl gene or reducing its activity is not complete, one would expect that there must be other important gcl targets. These targets could correspond to one or more of the other genes that are misexpressed in gcl PB/PGCs. Consistent with this possibility, transcriptional silencing in gcl PBs/PGCs is reestablished when terminal signaling is disrupted by mutations in the tsl gene. On the other hand, it is possible that excessive activity of the terminal signaling pathway also adversely impacts some non-transcriptional targets that are important for PB/PGC formation and that transcriptional silencing in only part of the story (see below) (Colonnetta, 2021).
Pae (2017) showed that mutations in the Gcl interaction domain of Torso (torsoDeg) stabilize the receptor and disrupt PGC formation. Consistent with the notion that Torso receptor is the primary, if not the only, direct target of gcl, they found that mutations in the Torso ligand modifier, tsl, or RNAi knockdown of torso rescued the PGC formation defects in gcl embryos. As would be predicted from those and the current findings, ectopic expression of the TorsoDeg protein induces the inappropriate transcription of sis-b and Sxl-Pe in PBs and newly formed PGCs. Thus, the failure to shut down ongoing transcription in gcl PBs and PGCs must be due (at least in part) to the persistence of the Torso receptor in the absence of Gcl-mediated degradation. Corroborating this idea, the ectopic activation of transcription in gcl PGCs is no longer observed when the terminal signaling pathway is disrupted by the removal of tsl. Taken together, these data strongly suggest that the establishment/maintenance of transcriptional silencing in PBs is a critical function of Gcl (Colonnetta, 2021).
Since RNAi knockdowns of terminal pathway kinases downstream of torso did not rescue gcl mutants, Pae (2017) postulated that the Tsl-Torso receptor interaction triggered a novel, non-canonical signal transduction pathway that disrupted PGC development. If that suggestion is correct, then the activation of sis-b and Sxl-Pe in PBs/PGCs in gcl and torsoDeg embryos would be mediated by this novel terminal signaling pathway. The results of the current study are ambiguous. Consistent with the suggestion of Pae, 2017, GOF mutations in MEK, a downstream kinase in the Torso signaling pathway, did not activate Sxl-Pe transcription in pole cells. However, an important caveat is that the GOF activity of MEK variants that was tested is likely not equivalent to the activity from the normal Torso-dependent signaling cascade. As the pole plasm contains at least two other factors that help impose transcriptional quiescence, the two GOF MEK mutants that were tested may simply not be sufficient to overcome their repressive functions. Two observations are consistent with this possibility. First, like torsoDeg, this study found that MEKE203K induces Sxl-Pe expression in male somatic nuclei. The same is true for a viable GOF mutation in Torso: it can induce ectopic activation of Sxl-Pe in male somatic nuclei, but is unable to activate Sxl-Pe in PGCs. Second, a key terminal pathway transcription target tailless is not expressed in gcl mutant PBs/PGSs even though the terminal pathway should be fully active. This is also true for embryos expressing torsoDeg and the two GOF MEK proteins. For these reasons, it cannot be unambiguously determined if it is the canonical terminal signaling pathway or another, noncanonical signaling pathway downstream of Torso that is responsible for the expression of sis-b, Sxl-Pe, and other genes in gcl mutant PB/PGCs (Colonnetta, 2021).
There are also reasons to think that the canonical Torso signal transduction cascade must be inhibited for proper PGC formation. One of the more striking phenotypes in gcl mutants is the dispersal of key germline mRNA and protein determinants into the surrounding soma. A similar disruption in the sequestration of pole plasm components is observed not only in torsoDeg embryos but also in MEKE203K and MEKF53S embryos. Thus, this gcl phenotype would appear to arise from the deployment of the canonical Torso receptor signal transduction cascade, at least up to the MEK kinase. However, this result does not exclude the possibility that the Tsl->Torso->ERK pathway has other non-transcriptional targets that, like Sxl-Pe expression, can also interfere with PB/PGC formation. If this was the case, it could potentially explain why global transcriptional inhibition failed to rescue the PGC defects in gcl embryos. In this respect, a potential-if not likely-target is the microtubule cytoskeleton. In previous studies, it was found that the PB and PGC formation defects as well as the failure to properly sequester critical germline determinants in gcl arise from abnormalities in microtubule/centrosome organization. Preliminary imaging experiments indicate that centrosome distribution of torsoDeg PBs is also abnormal, suggesting that inappropriate activation of the terminal signaling pathway perturbs the organization or functioning of the microtubule cytoskeleton and/or centrosomes. Such a mechanism would also be consistent with the dispersal of germline mRNA and protein determinants in torsoDeg and GOF MEK embryos. While further experiments will be required to demonstrate microtubule and centrosomal aberrations in torsoDeg and GOF MEK embryos, a role for a receptor-dependent MEK/ERK signaling cascade in promoting centrosome accumulation of γ-tubulin and microtubule nucleation has been documented in mammalian tissue culture cells. It is thus conceivable that MEK/ERK signaling has a similar role in Drosophila PB nuclei and PGCs. It will be important to determine if Torso-dependent activation of MEK/ERK can perturb the behavior or organization of centrosomes and/or microtubules in early embryos, and, if so, whether the influence can alter the pole plasm RNA anchoring and/or transmission. Taken together, the current data reveal a mutual antagonism between the determinants that specify germline versus somatic identity. Future studies will focus on how and when during early embryogenesis such feedback mechanisms are activated and calibrated to establish and/or maintain germline/soma distinction (Colonnetta, 2021).
Secretions within the adult female reproductive tract mediate sperm survival, storage, activation, and selection. Drosophila female reproductive gland secretory cells reside within the adult spermathecae and parovaria, but their development remains poorly characterized.
With cell-lineage tracing, this study found that precursor cells downregulate lozenge and divide stereotypically to generate three-cell secretory units during pupal development. The NR5A-class nuclear hormone receptor Hr 39 is essential for precursor cell division and secretory unit formation. Moreover, ectopic Hr39 in multiple tissues generates reproductive gland-like primordia. Rarely, in male genital discs these primordia can develop into sperm-filled testicular spermathecae. Drosophila spermathecae provide a powerful model for studying gland development. It is concluded that Hr39 functions as a master regulator of a program that may have been conserved throughout animal evolution for the production of female reproductive glands and other secretory tissues (Sun, 2012).
In species where fertilization takes place internally, including mammals and insects, a sperm's long and obstacle-filled journey through the female reproductive tract culminates in the penetration of the egg. Prior to reaching its target, both paternal and maternal reproductive tissues deploy mechanisms that strongly influence an individual sperm's chances for success. In particular, specialized glands in female reproductive tracts produce mucus-rich secretions that capacitate sperm to fertilize successfully, inhibit infection, and provide nutritional, maintenance, and storage factors. The interactions of sperm and seminal fluid with the female reproductive tract and its secretions in Drosophila offer an opportunity to genetically analyze these complex processes (Sun, 2012).
Two paired glands, spermathecae (SPs) and parovaria (POs), are the primary sources of secretions encountered by sperm within the Drosophila female reproductive tract (see Structure and origin of Drosophila female reproductive glands). Messenger RNAs (mRNAs) encoding serine proteases, serpins, antioxidants, immune proteins, and enzymes involved in mucus production are found in SPs. Whereas two SPs arise from the engrailed− (en−) and en+ domains of the A8 segment, both POs originate in the en+ domain of the A9 segment in the female genital disc during pupal development. Both types of mature gland contain large, polyploid secretory cells (SCs). Each SC connects with the gland lumen via a specialized cuticular canal equipped with a secretion-collecting 'end apparatus'. Anatomically related secretory units are found in SPs from other species and in insect epidermal glands that produce pheromones, venoms, and many other products. Despite their ubiquity, insect epidermal gland development has not been well characterized at the molecular genetic level (Sun, 2012).
Studies of genital disc development and patterning have identified multiple genes important for reproductive gland formation. lozenge (lz), encoding a runt-domain transcription factor, is essential for both SP and PO formation and may be directly regulated by the sex determination pathway. Homologous to mammalian AML-1, Lz also supports developing blood precursors and prepatterns ommatidial cells in the developing eye. The dachshund (dac) gene also acts in multiple imaginal discs and is specifically needed for spermathecal duct development. Mutations that disrupt sphingolipid metabolism also cause abnormalities in spermathecal number and structure (Sun, 2012).
One of the most interesting genes needed to form reproductive glands encodes the nuclear hormone receptor Hr39, an early ecdysone-response gene. Hr39 and Ftz-f1 are the only two NR5A class nuclear hormone receptors in Drosophila, a class that in mammals includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1). All four of these proteins share 60%-90% sequence identity within their DNA binding domains and bind in vitro to identical sequences. SF-1 is a master regulator of steroidogenesis and sex hormone production, whereas LRH-1 is required in the ovary for female fertility, in embryonic stem cells for pluripotency and in endodermal tissues for metabolic homeostasis. Weak Hr39 mutations alter the production of some SP gene products, whereas LRH-1 directly controls major secretory proteins of the exocrine pancreas. Thus, NR5A class hormone receptors may play a conserved role controlling secretions from certain tissues, including female reproductive glands (Sun, 2012).
This study characterized the cell lineage of developing reproductive glands and clarify the roles of lz and Hr39. Hr39 is expressed sex-specifically in lz-positive female gland primordia beginning shortly after the ecdysone pulse that initiates prepupal development. When levels of Hr39 are reduced, lz-expressing precursors fail to protrude, divide, or remain viable, suggesting that Hr39 expression orchestrates reproductive gland development. Mouse LRH-1, but not SF-1, can partially replace Hr39 function in gland formation. Ectopic expression of Hr39 in male larvae can induce a pigmented SP-like structure containing sperm to develop in the male reproductive tract. It is proposed that Hr39 acts as a master regulator of reproductive gland development and that the production of sperm-interacting proteins in the female reproductive tract under the control of NR5A proteins has been conserved during evolution. These findings suggest new targets for controlling agriculture pests and human-disease vectors (Sun, 2012).
These studies reveal that lz and Hr39, despite their nearly identical loss-of-function phenotypes, have distinctive expression patterns during gland development. All gland precursors express both genes following puparium formation, but within 24 hr divide to produce lz+ epithelial precursors apically and lz− SUPs basally. SUPs then differentiate according to a stereotyped program involving production of two transient accessory cells and a single polyploid secretory cell (Sun, 2012).
Reproductive secretory cells arise in a superficially similar manner to sensory bristles and multiple classes of mechanosensory and chemosensory sensilla. Both utilize short fixed-cell lineages that employ transient accessory cells to generate permanent extracellular structures (secretory canal, sensory bristle, etc.), but the three-cell secretory lineage analyzed in this study differs from the four asymmetric divisions producing five different cells typical of PNS differentiation (see Lineage Analysis of Secretory Unit Formation). Many other insect epidermal glands probably develop in a generally similar manner, but the precise cell lineages and mechanisms documented in this study for Drosophila reproductive glands (three cells, absence of ciliary involvement) differ from previous models (Sun, 2012).
Drosophila secretory units provide a powerful system for analyzing insect gland development. Studies in other insects suggested that an accessory cell utilizes a ciliary process to prevent the SCs from being sealed off by cuticle-secreting epithelial cells. This study found no morphological or genetic evidence that cilia are involved in forming Drosophila secretory units. However, the apical cell (AC) may fulfill this same role using normal microtubules, in much the same way that the anterior polar cells in egg chambers template the micropyle channel during oogenesis. Membranes from the basal cell (BC) likely surround this AC process, secrete the cuticular canal, and join it to the luminal cuticle. Concomitantly, the BC likely secretes the end apparatus around a large apical segment of the SC, which it surrounds (Sun, 2012).
The NR5A hormone receptor Hr39 plays multiple roles in reproductive gland development. Initially, Hr39 orchestrates gland protrusion and in the absence of Hr39 protrusion fails to occur. Among Drosophila imaginal discs, gland protrusion in genital discs is a unique process that leads to the differentiation of a gland capsule connected to the nascent reproductive tract by a tubular duct. When Hr39 is misexpressed, patches of cells within multiple imaginal discs that do not normally express Hr39 undergo changes reminiscent of early protrusion (Sun, 2012).
Hr39, a known member of the ecdysone response pathway, is likely to time reproductive gland cell divisions during pupal development. The initial Hr39 expression we observed in the genital disc was detected shortly after the prepupal ecdysone pulse. Several additional peaks of ecdysone titer during pupal development correspond closely with the timing this study measured of the secretory cell divisions. These observations suggest that external hormonal signals rather than internal autonomous mechanisms sometimes drive precise cell lineages. In addition to its requirement within cellular precursors, Hr39 mutations alter SP secretory gene mRNA levels (Allen, 2008), suggesting that Hr39 also regulates secretory gene expression within SCs (Sun, 2012).
Finally, Hr39 acts as a high level 'master regulator' by integrating individual pathways to elicit the production of an entire gland. Most cells expressing ectopic Hr39 could not progress past the initial stage of eversion, but in male genital discs Hr39-positive clones sometimes generated integrated structures that strongly resembled small spermathecae. They contained round heads with lumens, a pigmented layer, and rarely were connected to the male reproductive tract by ducts through which sperm were taken up. Thus, Hr39 (but not lz) can reprogram male genital cells to generate ectopic spermathecae that likely synthesize and secrete products attractive to sperm (Sun, 2012).
Drosophila reproductive gland development is unusually susceptible to perturbation. Rare adults in some wild strains contain an extra spermatheca, and females bearing weak alleles of either lz or Hr39 lose parovaria (POs) entirely and produce fewer spermathecae (SPs), which vary dramatically in size and cellular content. These effects probably result from the disparate sizes of the precursor pools for individual organs. PO pools are very small, whereas the exceptionally large posterior SP primordium may easily split in two under conditions where precursor proliferation is perturbed. The effects of dac mutations on duct structure are probably also due to altered precursor pools. Sphingolipids may affect gland development by serving as endogenous Hr39 ligands, consistent with reports that SF-1 can bind sphingolipids (Sun, 2012 and references therein).
In mammals, sperm interact with female secretory products at multiple locations. Glands within the uterine endometrium are hypothesized to govern selective passage through the cervix, uterus, and subsequently, the uterotubal junction. Following entry into the oviduct, sperm induce and interact with the products of specialized tubal secretory cells that likely mediate capacitation. In some species, these products also allow sperm to be stored in the oviduct while retaining their ability to fertilize an egg. Mammalian female reproductive glands continue to nurture preimplantation embryos and are likely essential for successful pregnancy (Sun, 2012).
Drosophila is emerging as a valuable model with which to study multiple aspects of reproductive physiology, some of which may have been conserved during evolution. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle (Simeone, 1995), but its role in fertility is unknown. The murine Hr39 homolog LRH-1 is required for female fertility, but whether it plays a role in reproductive gland secretion has yet to be tested. However, LRH-1 is required for the development of several exocrine tissues and in the pancreas is directly involved in the transcription of major secretory products. Thus, LRH-1 and Hr39 may both govern the formation and secretory function of exocrine tissue (Sun, 2012).
These study studies provide further support for the idea that an NR5a-dependent program of secretory cell development has been conserved in evolution. Murine LRH-1 can partially replace Hr39 function in Drosophila reproductive gland formation. Similar rescue with two other NR5A members (mammalian SF-1 or Drosophila Ftz-F1) failed and instead suppressed all gland formation. This is consistent with previous findings that Hr39 and Ftz-F1 have opposing roles in alcohol dehydrogenase and EcR expression. Antagonistic roles in gene regulation by the two NR5A family members may be evolutionarily conserved. Further study of the roles of Hr39 and LRH-1 should help define a fundamental program of secretory cell development that may be widely used (Sun, 2012).
Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca2+) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. This study provide the first evidence that Drosophila oocytes require Ca2+ for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. The results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca2+ to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca2+ and the activation of development (Horner, 2008).
Mature oocytes require an external signal to begin development. This signal, which differs among animals, 'activates' the oocyte to resume and complete meiosis, modify its outer coverings, reorganize its cytoskeleton, and translate or degrade certain maternal mRNAs. In most animals, activation is triggered by fertilization, but changes in the ionic environment, changes in pH, or mechanical deformation can initiate egg activation in some species. A frequent response to the activating trigger in vertebrates and marine invertebrates is a rise in free calcium within the egg. In these organisms calcium acts as a second messenger to drive the downstream processes of egg activation. In insects, the requirement for calcium during egg activation has never been directly tested; however, recent reports show that a calcium-responsive regulator, Sarah, is essential for egg activation in Drosophila melanogaster (Horner, 2006l; Takeo, 2006; Horner, 2008 and references therein).
Drosophila egg activation, as in other insects that have been examined, is independent of fertilization. Unfertilized laid eggs can complete meiosis, modify their vitelline membranes, and translate some maternal RNAs. Thus, Drosophila sperm trigger none of the traditional metrics of egg activation. Instead, activation initiates during ovulation but the activating signal itself remains unknown (Horner, 2008).
One hypothesis for the activating signal in Drosophila derives from studies of Hymenoptera in which embryo development is triggered by oviposition. It has been proposed that mechanical stress imparted upon the egg during passage through the ovipositor is the signal that starts development in Hymenoptera. For instance, in the haplodiploid wasp, Pimpla turionellae, the diameter of the ovipositor is about one-third of the width of the egg, suggesting that physical deformation during egg laying initiates development. Consistent with this hypothesis, when P. turionellae eggs are dissected from the ovary and squeezed through a narrow capillary tube, over 70% of eggs activate, as measured by their ability to develop into haploid male larvae. Pressure exerted on the egg can also activate eggs of another wasp, Nasonia vitripennis, since 23% of eggs dissected from the ovary and pressed with a needle were able to develop to larvae (Horner, 2008 and references therein).
The hypothesis that mechanical stimulation could also trigger Drosophila egg activation was initially suggested by two observations. First, it has been shown that application of hydrostatic pressure of an unspecified level or duration to Drosophila oocytes resulted in an increase in nuclear number in those oocytes. Whether such oocytes had properly completed meiosis and were undergoing haploid mitotic divisions was not reported. Second, it has been reported that pulling manually on the dorsal chorionic appendages of Drosophila oocytes triggered the resumption of meiosis in 3/3 cases. These intriguing observations suggested that mechanical stimulation might trigger the resumption and completion of meiosis, one aspect of egg activation. No other aspects of egg activation in response to mechanical stimulation were examined in those studies (Horner, 2008 and references therein).
Analogous to oviposition in wasps, a mechanical trigger might occur as Drosophila eggs move from the ovary into the narrow lateral oviduct during ovulation. Mechanical stimulation could rearrange egg contents, leading to new structural or molecular combinations. Alternatively, mechanical stimulation could stimulate a mechanically-gated (MG) process, such as the opening or closing of stretch-activated (SA) ion channels. Such alterations to ion channels could lead to ionic changes analogous to those that trigger egg activation in other metazoans (Horner, 2008).
Another potential activation trigger in Drosophila is hydration. Mature oocytes in the ovary appear desiccated, whereas laid eggs are taut and expanded. Some evidence suggests that the hydrated contents of the oviduct lumen are transferred to eggs during ovulation. Support for a hypothesis that hydration could lead to egg activation is that incubation in a hypotonic buffer in vitro causes oocytes to swell and activate. Such hypo-osmotic swelling in vivo or in vitro could serve as another form of mechanical stimulation, by altering membrane tension to trigger a MG process. Additionally, specific ion(s) in the hydrating medium could provide the activation signal (Horner, 2008).
To better understand the activating signal in Drosophila, whether pressure exerted on the egg effects activation was determined. It was found that external hydrostatic pressure accelerates activation, as assessed by vitelline membrane permeability changes and protein translation. In addition, an inhibitor of MG processes was able to inhibit hypo-osmotically induced activation, suggesting for the first time that the mechanism by which hydration leads to activation is through a MG response triggered by osmotic pressure. External calcium was shown to be necessary for both hypo-osmotic and pressure-accelerated activation. Therefore this study demonstrates that the phenomenon of calcium-dependent egg activation extends to a new and important class of metazoans: insects. Taken together, these results suggest that mechanical stimulation from hydration and/or physical deformation during ovulation triggers activation in Drosophila by causing an influx of calcium into the egg. Drosophila is now poised to join organisms traditionally used to study activation, with the advantage of valuable genetic resources to discover the likely conserved pathways that mediate egg activation (Horner, 2008).
Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. This study shows that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. The calcium rise required influx of calcium from the external environment and was induced as the egg ovulated. Pressure on the oocyte (or swelling by the oocyte) could induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts showed a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation required the IP3 system. Thus, although a fertilizing sperm was not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg was conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components (Kaneuchi, 2015).
Egg activation is a conserved phenomenon that prepares an animal oocyte for successful embryogenesis through completion of meiosis, restructuring of the vitelline membrane, and changes to the existing protein and mRNA pools within the egg. The trigger for Drosophila (and other arthropod) egg activation differs from the better-known cases of vertebrate and echinoderm egg activation in that it is decoupled from fertilization. Despite this critical difference in egg activation trigger mechanisms, this study reports that a calcium wave occurs during egg activation in Drosophila, as in other animals. In vivo imaging of oocyte calcium levels indicates that the intracellular calcium rise is triggered by ovulation. In vitro imaging shows that this rise takes the form of wave(s) that initiate from egg pole(s) and move across the egg; the rise in cytosolic calcium is then followed by a decrease. It is proposed that this dynamic rise and fall in cytosolic calcium triggers the events of egg activation in Drosophila, as suggested for the calcium transients in other organisms such as mouse (Kaneuchi, 2015).
The calcium rise during Drosophila egg activation can occur only in the presence of calcium in the extracellular environment. It is proposed that the wave initiates when Ca2+ enters the oocyte through activation of mechanosensitive ion channels on the oocyte cell surface. These channels are proposed to be activated by either or both of the following mechanisms. First, the oocyte swells as it passes through the oviducts, presumably by taking up fluid: mature oocytes in the ovary are shriveled in appearance, but laid eggs are swollen and taut. In in vitro experiments, it was noted that the calcium wave does not initiate until after the egg has begun to swell. Additionally, it was possible to increase the speed of initiation by adding a few drops of water to the activating medium during imaging, thus increasing hypotonicity and causing faster egg swelling. It is postulated that swelling exerts a stretch tension force on the membrane, which triggers the opening of mechanosensitive Ca2+ channels. Second, it was shown that, independently of oocyte swelling, mechanical pressure exerted on the oocyte is capable of initiating the wave. It is proposed that oocytes may be subjected to both triggers during ovulation: pressure from the outside as they move out of the ovary and into the oviducts and swelling as they encounter the oviductal fluid. As a result, mechanosensitive ion channels open, and calcium levels rise in the oocyte. In vivo imaging of oocytes as they are ovulating supports the pressure hypothesis: the movement of the oocyte into the oviduct is not smooth and fluid; instead, the oocyte moves slowly at first and then rather suddenly pushes into the oviduct, as if it meets some resistance force as it begins ovulation (Kaneuchi, 2015).
Recent evidence from mice indicates that a requirement for external Ca2+ for egg activation is not unique to insects like Drosophila, although a requirement for calcium influx to initiate the first wave is. In mice, after the initial Ca2+ rise induced by sperm PLC, further calcium oscillations require Ca2+ uptake from the extracellular environment through a store-operated Ca2+ entry mechanism; when intracellular ER Ca2+ stores are depleted, plasma membrane channels open to allow Ca2+ back into the cell (Kaneuchi, 2015).
How could a wave be triggered from the egg pole(s)? It is possible that the mechanosensitive ion channels that mediate the calcium rise are localized at the poles, analogous to the localization of some of the embryo-polarity machinery including a terminal-group signaling cascade that marks the two ends of the embryo as similar to one another but different from the interior. In this model, mechanical cues applied to the egg would activate those channels, and because they are at the poles, the wave would initiate at the poles. It will be intriguing to test this hypothesis by determining which mechanosensitive ion channels are needed to trigger the calcium wave (and egg activation) and whether they show polar localization. Toward this end, the finding that the wave is inhibited by gadolinium and ACA suggests that the relevant channels might be members of the TRP family of calcium channels. The best candidates are three TRP family channels that are expressed in the ovary [painless (TRPA1), trpm (TRPM3), and trpml (TRPP1/Pkd2)]. Further experiments will be needed to determine the particular channel(s) that is needed to initiate the wave and its localization in the oocyte membrane. Alternatively, it is possible that the required channels are not localized but rather that the egg cytoskeleton is less rigid on the poles of the egg. Normally, uniform swelling of a prolate spheroid (such as a Drosophila oocyte) would exert greater tension along the center or waistline. However, different cytoskeletal makeup at the poles may cause tension to be experienced differently there, and in this way channels spread uniformly throughout the plasma membrane may open first at the poles. Further experiments will be required to determine why the wave initiates at the poles (Kaneuchi, 2015).
In other organisms in which the signaling pathway has been studied downstream of the Ca2+ influx, an increase in intracellular Ca2+ is thought to be the ultimate cause of the meiosis resumption that permits subsequent embryonic mitosis, and of changes in macromolecular synthesis or stability. However, the way in which these events are connected to the calcium wave is still unclear in any system. This study has shown that a calcium wave occurs during Drosophila egg activation and that the sensitivity of this wave to manipulations (pressure, swelling, chemical inhibitors) mirrors that for egg activation events. Given this finding, and the fact that many signaling pathways and events downstream of the calcium signal appear to be conserved between Drosophila other species, Drosophila will offer the unique perspective of isolating
egg activation events from fertilization events, as well the possibility of genetic manipulation and larger-scale ‘omics' studies that will help to link a Ca2+ flux to downstream egg activation events (Kaneuchi, 2015).
Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal (Sperling, 2023).
The endosymbiotic bacteria Wolbachia pipientis is known to infect a wide range of arthropod species yet less is known about the coevolutionary history it has with its hosts. Evidence of highly identical W. pipientis strains in evolutionary divergent hosts suggests horizontal transfer between hosts. For example, Drosophila ananassae is infected with a W. pipientis strain that is nearly identical in sequence to a strain that infects both D. simulans and D. suzukii, suggesting recent horizontal transfer among these three species. However, it is unknown whether the W. pipientis strain had recently invaded all three species or a more complex infectious dynamic underlies the horizontal transfers. This study examined the coevolutionary history of D. ananassae and its resident W. pipientis to infer its period of infection. Phylogenetic analysis of D. ananassae mitochondrial DNA and W. pipientis DNA sequence diversity revealed the current W. pipientis infection is not recent. In addition, the population genetics and molecular evolution of several Germline Stem Cell (GSC) regulating genes of D. ananassae were examined. These studies reveal significant evidence of recent and long-term positive selection at stonewall in D. ananassae, while pumillio showed patterns of variation consistent with only recent positive selection. Previous studies had found evidence for adaptive evolution of two key germline differentiation genes, bag of marbles (bam) and benign gonial cell neoplasm (bgcn) Maternally inherited intracellular bacteria Wolbachia cause both parasitic and mutualistic effects on their numerous insect hosts that include manipulating the host reproductive system in order to increase the bacteria spreading in a host population, and increasing the host fitness. This study demonstrates that the type of Wolbachia infection determines the effect on Drosophila melanogaster egg production as a proxy for fecundity and metabolism of juvenile hormone (JH), which acts as gonadotropin in adult insects. This study used six D. melanogaster lineages carrying the nuclear background of interbred Bi90 lineage and cytoplasmic backgrounds with Wolbachia of different genotype variants or without it. wMelCS genotype of Wolbachia decreases the egg production in the infected D. melanogaster females in the beginning of oviposion and increases it later (since the sixth day after eclosion), wMelPop Wolbachia strain causes the opposite effect, while wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on these traits compared to uninfected Bi90 D. melanogaster females. The intensity of JH catabolism negatively correlates with the fecundity level in the flies carrying both wMelCS and wMelPop Wolbachia The JH catabolism in females infected with genotypes of wMel group does not differ from that in uninfected females. The effects of wMelCS and wMelPop infection on egg production can be leveled by the modulation of JH titre (via precocene/JH treatment of the flies). Thus, at least one of the mechanisms, promoting the effect of Wolbachia on D. melanogaster female fecundity, is mediated by JH (Gruntenko, 2019).
Cytoplasmic incompatibility (CI) is the most common phenotype induced by endosymbiont Wolbachia and results in embryonic lethality when Wolbachia-modified sperm fertilize eggs without Wolbachia. However, eggs carrying the same strain of Wolbachia can rescue this embryonic death, thus producing viable Wolbachia-infected offspring. Hence Wolbachia can be transmitted mainly by hosts' eggs. By RNA-seq analyses, the transcription profiles of Drosophila melanogaster adult ovaries were first compared with and without the wMel Wolbachia, and 149 differentially expressed genes (DEGs) were identified, of which 116 genes were upregulated and 33 were downregulated by Wolbachia infection. Seven microRNAs (miRNAs) were identified that were all upregulated in fly ovaries by Wolbachia infection. Matching of miRNA and mRNA data showed that these seven miRNAs regulated 15 DEGs. Most of the DEGs showed variation in opposite directions in ovaries versus testes in the presence of Wolbachia, which generally supports the "titration-restitution" model for CI. Furthermore, genes related to metabolism were upregulated, which may benefit maximum proliferation and transmission of Wolbachia. This provides new insights into the molecular mechanisms of Wolbachia-induced CI and Wolbachia dependence on host ovaries (He, 2019).
In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led to a hypothesis that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, the specificity of the genetic interaction between bam and W. pipientis must be understood. This study used CRISPR/Cas9 to engineer the original single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. The fertility was assessed of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/bamL255F mutant females, each infected individually with 10 W. pipientis wMel variants representing three phylogenetic clades. Overall, it was found that all of the W. pipientis variants tested rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. In addition, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype (Bubnell, 2021).
The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).
After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).
By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions inluence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).
Tissues use numerous mechanisms to change shape during development. The Drosophila egg chamber is an organ-like structure that elongates to form an elliptical egg. During elongation the follicular epithelial cells undergo a collective migration that causes the egg chamber to rotate within its surrounding basement membrane. Rotation coincides with the formation of a 'molecular corset', in which actin bundles in the epithelium and fibrils in the basement membrane are all aligned perpendicular to the elongation axis. This study shows that rotation plays a critical role in building the actin-based component of the corset. Rotation begins shortly after egg chamber formation and requires lamellipodial protrusions at each follicle cell's leading edge. During early stages, rotation is necessary for tissue-level actin bundle alignment, but it becomes dispensable after the basement membrane is polarized. This work highlights how collective cell migration can be used to build a polarized tissue organization for organ morphogenesis (Cetera, 2014).
Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. This study used live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber's epithelial cells and undergo oriented insertion into the BM by directed epithelial migration. It was found that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, it was shown that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape (Isabella, 2016). Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers are comprised of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a "molecular corset" mechanism, wherein structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. This study provides evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ (Andersen, 2016).
It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. This paper shows that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. It was also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation (Huang, 2014).
Many adult stem cell communities are maintained by population asymmetry, where stochastic behaviors of multiple individual cells collectively result in a balance between stem cell division and differentiation. This study investigated how this is achieved for Drosophila Follicle Stem Cells (FSCs) by spatially-restricted niche signals. FSCs produce transit-amplifying Follicle Cells (FCs) from their posterior face and quiescent Escort Cells (ECs) to their anterior. JAK-STAT pathway activity, which declines from posterior to anterior, dictates the pattern of divisions over the FSC domain, promotes more posterior FSC locations and conversion to FCs, while opposing EC production. Wnt pathway activity declines from the anterior, promotes anterior FSC locations and EC production, and opposes FC production. The pathways combine to define a stem cell domain through concerted effects on FSC differentiation to ECs and FCs at either end of opposing signaling gradients, and impose a pattern of proliferation that matches derivative production (Melamed, 2020).
Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, two additional developmental amplicons, termed DAFC (Drosophila Amplicon in Follicle Cells)-30B and -62D were identified in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression (Claycomb, 2004).
The maximally amplified genes in DAFC-62D, yellow-g and yellow-g2, are members of the yellow gene family that are predicted to encode secreted proteins. The family shares homology with the Major Royal Jelly Protein Family in honeybees (Apis mellifera), involved in the specification of the queen bee. The founding member of the Yellow family, Yellow-y, is known to play a role in mating behavior and in the melanization and hardening of the adult cuticle. Other Yellow family members have been shown to act as dopachrome-conversion enzymes that catalyze a key reaction in the melanization process. Interestingly, a similar process is used in the hardening of the egg chorion in mosquitoes and suggests that Yellow-g and Yellow-g2 may play a catalytic role in the crosslinking of the chorion and/or underlying vitelline membrane proteins in Drosophila (Claycomb, 2004).
A second group of genes encodes proteins with chitin binding motifs that could function in egg production. Genes of this type are present in both amplicons, with DAFC-62D containing two such genes and DAFC-30B containing one. Chitin binding domains serve an antimicrobial function in a variety of plants and marine invertebrates. Homologs of marine invertebrate proteins, such as tachycitin, could provide the egg with protection against microbes. Alternatively, chitin, a structural polysaccharide found in many organisms, could also be a component of the eggshell, and interaction with the chitin binding proteins might contribute to eggshell integrity (Claycomb, 2004).
In both DAFC-30B and 62D, there are also a number of genes whose role in follicle cells is not yet clear. These include both genes encoding proteins without known sequence motifs and genes whose products are predicted to have the enzymatic activities of adenylate cyclases, membrane transporters, calcium-transporting ATPases, GTP dissociation inhibitors, and others (Claycomb, 2004).
The yellow-g gene is essential for a rigid eggshell, and the predicted gene products of the yellow-g and yellow-g2 genes suggest a molecular explanation for these mutant defects. The eggshell is composed of several layers, including the outermost exochorion, the endochorion, the inner chorion layer, and the vitelline membrane, which is the innermost structure that also contacts the oocyte. The collapsed embryos and disrupted vitelline membranes that result from mutation of yellow-g indicate that yellow-g is necessary for the structural integrity of the eggshell. At the level of the light microscope, the exochorion of embryos laid by mutant mothers appears normal. The collapsed embryos are reminiscent of vitelline membrane defects, leading to the hypothesis that yellow-g is necessary for proper vitelline membrane formation (Claycomb, 2004).
It is proposed that Yellow-g and Yellow-g2 act to crosslink the vitelline membrane, or perhaps the inner chorion layer. The Yellow family members, Yellow-f and Yellow-f2, are capable of catalyzing the conversion of dopachrome to dihydroxyindole, a limiting step in the melanization pathway, during larval, pupal, and adult stages. The enzymatic events leading to the crosslinking of the vitelline membrane are not well understood, but seem to involve one phase of disulfide bond formation and a subsequent disulfide bond-independent phase. Additionally, the α methyl dopa resistant (amd) gene product, which acts in the conversion of dopamine during the polymerization of the adult cuticle, is required in the follicle cells for proper vitelline membrane crosslinking. This suggests that a similar set of dopamine conversion reactions catalyzed by Yellow-g and Yellow-g2 may be necessary for the crosslinking of the vitelline membrane just prior to egg laying. Consistent with this hypothesis, it is observed that eggs laid by homozygous yellow-g mutant females are highly sensitive to sodium hypochlorite (bleach), and the majority of these embryos burst upon brief exposure. Of the remaining, intact embryos, 100% were permeable to the dye neutral red, which has been used to assay vitelline membrane defects. These results are indicative of a failure to crosslink the vitelline membrane and further implicate yellow-g in the crosslinking process. However, this hypothesis does not explain the specific expression of the yellow-g and yellow-g2 genes in the follicle cells producing the micropyle late in egg chamber development. It is possible that crosslinking of the vitelline membrane or inner chorion layer within this specialized structure requires distinct regulation or timing. A more detailed analysis of the eggshell defect and biochemical studies of Yellow-g and Yellow-g2 will help gain a better understanding of the steps necessary for vitelline membrane crosslinking and will uncover any specialized micropyle functions (Claycomb, 2004).
DAFC-30B and DAFC-62D provide insights into the use of amplification as a developmental strategy. All of the previously characterized amplified genes play a purely structural role in eggshell formation; no enzymes necessary for proper eggshell formation have been examined. None of the genes of DAFC-30B and DAFC-62D encode known structural components of the eggshell. However, several of the amplified genes that are highly expressed in follicle cells, including CG18419 and the yellow-g genes, encode products predicted to possess enzymatic, signal transduction, or transporting activities. Furthermore, at least yellow-g is essential for proper egg formation, thus revealing an additional function of amplification: to increase the levels of enzymes needed to catalyze developmentally important reactions. Thus the identification of additional amplicons highlights genes likely to be crucial in developmental events and opens the possibility that other tissues employ amplification to maximize gene expression during differentiation. It is surprising that a 4- to 6-fold increase in gene copy number would affect gene product levels in a developmentally significant manner. It is possible, however, that copy number increases are considerably higher in subsets of follicle cells, or that the replication process itself facilitates transcription (Claycomb, 2004).
The follicle cell amplicons serve as superb model metazoan replicons, permitting delineation of cis-regulatory elements, identification of replication proteins, and clarifying the developmental control of the initiation and elongation. Developmental distinctions between DAFC-62D and the previously studied DAFCs provide clues into how origin firing can be linked to developmental signals. It has been shown by real-time PCR that replication initiates at DAFC-66D and -7F, coupled with replication fork movement, during egg chamber stages 10B and 11. Subsequently (stages 12 and 13), origins cease firing and only existing replication forks move bidirectionally to produce a gradient of copy number that extends over 100 kb. Furthermore, the replication initiation factor ORC2 localizes to amplification origins only during the initiation phase and dissociates at the onset of the elongation phase. Replication factors involved in multiple steps of DNA replication, such as MCM2-7 and PCNA, colocalize with BrdU throughout amplification (Claycomb, 2004).
DAFC-62D behaves differently from these amplicons and from DAFC-30B. There is a final increase in copy number at a very precise region of the amplicon, about 1.5 kb downstream of yellow-g2, during stage 13. As it is the peak of amplification, this region is likely to possess a replication origin. Understanding how DAFC-62D can undergo a final initiation hours after ORC is no longer detectable at origins by immunofluorescence will provide insights into the control of replication initiation. The additional replication in stage 13 may occur in only subsets of follicle cells, and ORC could persist specifically at DAFC-62D in these cells. For example, additional gene copies could permit optimal levels of expression of the yellow-g genes in the follicle cells building the micropyle (Claycomb, 2004).
These studies were initiated to devise a systematic approach for finding developmental amplicons. The microarray assay is sensitive and can detect low levels of gene amplification, and amplification levels as low as 4-fold can be developmentally important. Thus, this approach will be invaluable in surveying for gene amplification in a number of tissues and in a variety of organisms where amplification has not been detected. Not only has the microarray strategy identified additional amplicons, but when coupled with the power of a genetic organism, it has proven to be a functional genomics approach for highlighting genes involved in specific developmental pathways (Claycomb, 2004).
Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. The activation of origins responsible for developmental gene amplification was examined in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. This study examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. It is proposed that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC (Liu, 2015).
Replication origins are under tight regulation to ensure activation occurs only once per cell cycle. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions. It has been proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks. This study utilized the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control, to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs). Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation. This study shows that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation (Alexander, 2015).
Dorsal appendage morphogenesis in Drosophila oogenesis has been used as a model system for studying the
relationship between patterning and morphogenesis. Each
of the two dorsal respiratory appendages of the Drosophila
egg chamber is formed by secretion of eggshell proteins into
a tube of follicle cells. This tube is generated by cell shape
changes and rearrangements within an epithelial sheet. Dorsal appendage
formation is therefore similar to more complicated
examples of organogenesis. In addition, the study of dorsal
appendage formation provides several advantages that make
it an excellent system for investigating the regulation of
epithelial morphogenesis. For example, the signaling events that
determine two populations of dorsal follicle cells are well
understood. This understanding facilitates an ability to uncouple effects on patterning from morphogenesis. Further, powerful genetic tools, including
mutations that disrupt dorsal appendage formation, have
allowed for an unraveling of the genetic circuitry underlying
the regulation of epithelial morphogenesis (French, 2003).
The Drosophila egg chamber contains 16 interconnected
germline cells, consisting of 1 oocyte nourished by 15
highly polyploid nurse cells; these germline cells are surrounded
by a monolayer of ~1000 somatic follicle cells. The follicle cells secrete
the chorion that makes up the three layers of the eggshell:
the vitelline envelope, the endochorion, and the exochorion.
A subset of these follicle cells undergoes morphogenesis
to generate the dorsal appendages, specialized structures
that facilitate gas exchange in the developing embryo (French, 2003).
At stage 10 of oogenesis, the oocyte occupies the posterior
half of the egg chamber, the nurse cells the anterior
half, and the oocyte nucleus is positioned at the dorsal
anterior corner of the oocyte. The majority of follicle cells
forms a columnar layer over the oocyte, while a few follicle
cells are stretched out over the nurse cells. During stage
10B, those follicle cells closest to the nurse cell/oocyte
boundary begin to migrate centripetally, between nurse
cells and oocyte. The centripetal cells secrete the operculum (a thin layer of chorion that functions as an
escape hatch for the larva), the collar (a hinge on
which the operculum swings), and the micropyle, a coneshaped
structure through which the sperm enters (French, 2003).
Shortly after centripetal migration (stage 10B), the nurse
cells rapidly transfer their contents into the oocyte (stage
11) then begin to degenerate and undergo apoptosis (stages
12-14). At the same time, two groups of approximately
65-80 anterior, dorsal follicle cells, one on each side of the
dorsal midline of the egg chamber, migrate over the nurse
cells, laying down the chorion of the two dorsal appendages.
Extensive studies have defined the signaling events that
determine two populations of dorsal follicle cells. Dorsal
follicle-cell fate determination begins when transcripts encoding
the TGFalpha-like signaling molecule Gurken (Grk)
become localized in a cap above the oocyte nucleus. Grk
signals via the epidermal growth factor receptor homolog
(Egfr) to the follicle cells, activating a signal transduction
cascade involving the Ras/Raf/MAPK pathway. This initial signaling event defines a set
of dorsal anterior follicle cells and induces a second signaling
cascade involving three additional Egfr ligands. This
second cascade amplifies and refines the initial Grk signal,
leading to the definition of two separate populations of
dorsal follicle cells. These events are required for the production
of two separate dorsal appendages. Disruptions of this
process result in dorsalization or ventralization of the
follicular epithelium and the eggshell. Partial ventralization
generally results in failure to determine two separate
populations of cells, leading to the production of a single
dorsal appendage at the dorsal midline. Complete ventralization
results in the absence of dorsal cell fates and the
concomitant loss of dorsal appendages (French, 2003).
Information along the anterior-posterior axis also contributes
to cell-fate determination within the dorsal appendage
primordia. The BMP2/4 homolog encoded by
dpp is expressed in the stretch cells and a single row of
centripetally migrating cells. This morphogen radiates posteriorly
and alters columnar cell fates. High levels of Dpp repress dorsal identities and specify operculum; moderate levels synergize with Grk to
define dorsal, while low levels of Dpp are insufficient to
allow cells to respond to Egfr signaling. Thus, loss-of-function mutations generate short, often paddleless appendages, while overexpression either
expands the operculum at the expense of appendage material
or creates multiple, often antler-shaped dorsal structures.
The subsequent events underlying dorsal appendage morphogenesis
are only beginning to be understood. Analyses of
cultured wild-type egg chambers have revealed several
phases of dorsal appendage morphogenesis. From stages 10B to 12, two
groups of dorsal anterior follicle cells move out from the
follicular epithelium to form short tubes. Each tube extends
forward over the nurse cells, secreting chorion proteins that
make up the cylindrical stalk of the dorsal appendage. Cells at the anterior end of the tube change shape to
produce the flattened paddle of the distal dorsal appendage. Finally, upon oviposition, the entire follicular epithelium sloughs off, leaving behind the chorionic structures (French, 2003).
Elevated levels of human chitinase-like proteins (CLPs) are associated with numerous chronic inflammatory diseases and several cancers and can promote disease progression by remodeling tissue, activating signaling cascades, stimulating proliferation and migration, and by regulating adhesion. This study has identified Imaginal disc growth factors (Idgfs), orthologs of human CLPs CHI3L1, CHI3L2, and OVGP1, in a proteomics analysis designed to discover factors that regulate tube morphogenesis. The approach used magnetic beads to isolate a small population of specialized ovarian cells, cells that non-autonomously regulate morphogenesis of epithelial tubes that form and secrete eggshell structures called dorsal appendages. Elevated levels were detected of four of the six Idgf family members (Idgf1, Idgf2, Idgf4, and Idgf6) in flies mutant for Bullwinkle, which encodes a transcription factor and is a known regulator of dorsal-appendage tube morphogenesis. During oogenesis, dysregulation of Idgfs (either gain or loss of function) disrupts the formation of the dorsal-appendage tubes. Previous studies demonstrate roles for Drosophila Idgfs in innate immunity, wound healing, and cell proliferation and motility in cell culture. This study identified a novel role for Idgfs in both normal and aberrant tubulogenesis processes (Zimmerman, 2017).
Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. One daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis (Nystul, 2010).
The Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell. New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (see Prefollicle cells associate with cysts in an ordered fashion downstream from follicle stem cells). Follicle formation begins in the germarium, a structure at the tip of each ovariole that houses 2-3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches. Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte. At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a 'lens shape' characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Nystul, 2010).
A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning. However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs, low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Nystul, 2010).
The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production, and depends on Notch signals received from the germline. Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells. While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b. In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2. Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5. Moreover, polar and stalk are believed to arise exclusively from 'polar/stalk' precursors that separate from the rest of the FSC lineage and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Nystul, 2010).
This study analyzes the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and it was shown that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. These results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution (Nystul, 2010).
The data provide a much clearer picture of the follicle stem cell lineage than was previously available. They suggest that key aspects of FSC regulation depend on mechanisms that move cysts into a single file and program the loss of their escort cells precisely as they encounter FSCs and enter region 2b. Contact with incoming region 2a cysts likely induces FSC divisions, ensuring that cysts acquire a daughter cell from each stem cell as they stretch out to span the width of the germarium at the region 2a/2b junction. The asymmetry in cyst organization exposes FSC daughter cells to different cyst faces and, therefore, potentially to different signals. The FSC and daughter located on the same side as the entering cyst are exposed to the posterior face of the cyst while it is still in region 2a, covered by escort cells. In contrast, the opposite FSC and daughter contact the anterior face of the cyst as it migrates into 2b, at a time when the cyst is shedding its escort cell layer and exposing the Delta signal on the germ cell surface. Since region 2a cysts tend to interdigitate in forming a single file, cyst entry will usually alternate sides as successive cysts pass, causing FSC daughters arising from the same side to alternate migration paths. An advantage to this system may be its flexibility, allowing follicles to form normally even if multiple cysts enter from the same side in succession (Nystul, 2010).
Notch signaling in early FSC daughters promotes a 'prefollicle' state by blocking follicle cell differentiation. Consistent with this, it was observed that FSCs and their early daughters have much lower levels of differentiation markers such as FasIII and IMP-GFP. This developmental delay may prevent prefollicle cells from immediately incorporating into the differentiated follicular epithelium, allowing them to instead retain a more mesenchymal character conducive to cross-migration, and may also contribute to their ability to compete with the resident FSC for niche occupancy. Notch mutant daughters did not replace wild-type FSCs, most likely because they were unable to migrate into proximity. A role for Notch in suppressing differentiation downstream from the FSC might also explain why cells expressing activated Notch failed to migrate posteriorly (Nystul, 2010).
Follicle cell fates are specified by intercellular signals rather than lineage:
The two FSC daughters and their descendants, with few exceptions, continue to associate with the cyst they first contact at the 2a/2b boundary throughout subsequent development. Their division rate increases briefly, because daughters divide four times in the time it takes to generate three new cysts. Despite their growing number, however, all the cells retain the ability to produce main body, stalk, and polar cells for at least the first two to three divisions (8- to 16-cell stage). In contrast to previous reports, no evidence was found that polar and stalk cells derive from a lineage-restricted polar/stalk precursor population. Claims of a polar/stalk fate were based on experiments using higher rates of clone induction than in the experiments reported in this study. While many clones were also observed in these studies that contained both polar and follicle cells or both stalk and follicle cells, they were discounted as double clones (Nystul, 2010).
By examining clones induced at low frequency (more than threefold lower than in previous studies) it was possible to minimize the need for statistical correction for double clones. Furthermore, by studying clones induced at multiple times downstream from the FSC, overweighting small clones induced just as the polar and stalk cell fates are being determined by signaling within small cell groups was avoided. This has the effect of increasing the proportion of clones containing only one or two cell types even in the absence of any lineage restriction. At early, intermediate, and late times in somatic cell development in the germarium, clones that included all combinations of cell fates were always observed, indicating that follicle cells are multipotent prior to polar or stalk specification. This fits well with recent studies showing that many additional cells in the germarium can be induced to take on a polar cell fate by strong Notch signaling, while high levels of JAK-STAT signaling can induce more stalk cells. In contrast, no mechanism, time, or location where putative polar/stalk precursor cells are specified has ever been documented. Previous models also did not explain how these cells would preferentially arrive in the zone of cells separating regions 2b and 3 or what would become of the many extra cells that can sometimes be found in this region beyond the number needed for these fates (Nystul, 2010).
The finding that polar cells are initially specified in region 2b suggests that more spatial information is available within region 2b follicles than has been detected in earlier experiments. It was found that the first anterior and posterior polar cells are specified when cysts are associated with 8- to 16-cell follicle cells, in mid-to-late region 2b. This agrees closely with previous studies, which found that polar cells were first specified at the 14-cell stage. The early polar cells are detected by lineage because they cease dividing; however, no gene expression markers specific for these cells have been identified. Consequently, it remains uncertain where they are located at the time of induction or whether they function while remaining in region 2b. Since evidence was observed of Notch signal reception within individual follicle cells located at the anterior and posterior regions of stage 2b cysts, the simplest model is that these polar cells are induced by Delta signaling from the germline in a normal anterior/posterior (A/P) orientation. Although no Upd expression was detected at this time, these cells may nonetheless signal to the surrounding somatic cells to establish the graded levels of cadherin that define the initial anterior/posterior axis of the cyst (Nystul, 2010).
Where does the information come from that allows a small number of polar cells to be specified at this time? One possibility is a 'signal relay' from more posterior follicles. Highly accurate timing of polar cell formation relative to the signaling events during follicle budding might help to further test this model. However, the observation of localized Notch signal reception and polar cell specification in region 2b follicles suggests that the germline at this stage is already sufficiently polarized to signal in a limited manner along the future A/P axis. Some of this information may come from the inherent asymmetry within the germline cyst whose cells differ systematically in their fusome content, organelle content, and microtubule organization. The future oocyte and its sister four-ring canal cell are always located in the center of the region 2b cysts and hence might be one source for this inductive signal. Alternatively, there may be additional differences within this region of the germarium that have yet to be detected and that may contribute (Nystul, 2010).
These studies confirm previous conclusions that additional polar cells are formed during the process of budding and provide new insight into the budding process itself. Anterior-biased clones were almost always confined to a single follicle, but a significant fraction of posterior-biased clones (~33%) extended onto the next older follicle where they encompassed both an anterior polar cell and 2-30 anterior follicle cells. This suggests that cells at the posterior of the nascent follicle outgrow their cyst as it rounds up and are forced into the space between the posterior 2b cyst and the budding cyst. The origin of these cells has long been a mystery. A fraction of the interstitial cells likely contact and move onto the anterior of the downstream cyst where those that happen to lie adjacent to the existing polar cells are induced as new polar cells and stalk cells. Any remaining interstitial cells likely rejoin the main body of follicle cells as budding is completed or are eliminated by apoptosis as the stalk resolves to its final size (Nystul, 2010).
This study of early follicle cell development provides a rare opportunity to analyze how epithelial cells behave downstream from a stem cell. Most characterized Drosophila stem cell daughters receive information asymmetrically from their mother stem cell and differentiate rapidly. Germline stem cells and their niches ensure that cystoblasts receive an asymmetric fusome segment as well as differential environmental signals that program exactly four stereotyped divisions prior to entering meiosis. Under nonstressed conditions, intestinal stem cells utilize Notch signals to specify their daughters as either enterocytes or enteroendocrine cells and to terminate subsequent division. Neuroblasts program a stereotyped sequence of daughter cell fates by differential division and signaling. In contrast, FSC daughters undergo eight to nine divisions and differentiate independently of lineage over the course of several divisions and are capable of producing normal follicles even when the usual pattern of cellular interactions is altered. The increased resolution of follicle cell behavior afforded by these studies provides a valuable opportunity to study how epithelial cells are able to robustly bring about defined outcomes in the absence of the precise early programming (Nystul, 2010).
Several mechanisms are likely to contribute to successful follicle formation. First, genes characteristic of a polarized epithelium turn on slowly downstream of the FSC. The cross-migrating cell and several other cells frequently lacked such gene expression, but instead expressed genes characteristic of escort cells, suggesting that follicles are able to maintain some germline-soma interactions while completely replacing their somatic coverings. The early differentiation of polar cells may help guide subsequent cell behavior. In conjunction with the intrinsic asymmetric structure of germline cysts, differential adhesive interactions between germ and somatic cells across the follicle, differential pressures resulting from cell growth, and the resistive forces of the ovariolar wall, signals from these cells may be sufficient to ensure that the oocyte moves to the posterior and that cysts begin to round (Nystul, 2010).
These characteristics of the FSC lineage, although unique among well-studied stem cells in Drosophila, may be closer to those governing the epithelial lineages within many mammalian tissues. Thus, the mechanisms that give FSCs and their daughters their developmental flexibility and robustness are likely to be both widespread and medically relevant (Nystul, 2010).
Cell migration within a natural context is tightly controlled, often by specific transcription factors. However, the switch from stationary to migratory behavior is poorly understood. Border cells perform a spatially and temporally controlled invasive migration during Drosophila oogenesis. Slbo, a C/EBP family transcriptional activator, is required for them to become migratory. Wild-type and slbo mutant border cells as well as nonmigratory follicle cells were purified and comparative whole-genome expression profiling was performed, followed by functional tests of the contributions of identified targets to migration. About 300 genes were significantly upregulated in border cells, many dependent on Slbo. Among these, the microtubule regulator Stathmin was strongly upregulated and was required for normal migration. Actin cytoskeleton regulators were also induced, including, surprisingly, a large cluster of 'muscle-specific' genes. It is concluded that Slbo induces multiple cytoskeletal effectors, and that each contributes to the behavioral changes in border cells (Borghese, 2006).
Only one of the identified cytoskeletal regulators is known to affect microtubules, namely, Stathmin. Mammalian Stathmin/Op18 protein is well characterized. It binds to microtubules and promotes depolymerization by sequestration of tubulin dimers or direct action at microtubule ends. Interestingly, the activity of Stathmin can be regulated by phosphorylation in response to signaling or cell cycle phases. Drosophila Stathmin appears to have similar biochemical features. The availability of an antibody directed against Drosophila Stathmin allowed analysis of protein levels in situ. As expected, the level of Stathmin was higher in border cells than follicle cells. When analyzing slbo mutant border cells, a clear difference was observed between the inner polar cells and the outer border cells. The outer border cells are the migratory cells and require Slbo expression. In these cells, Stathmin expression was undetectable in the absence of Slbo, indicating a very strong dependence on Slbo. In contrast, Stathmin was still expressed in mutant polar cells, explaining why only a moderate reduction of stathmin mRNA levels was seen in whole border cell clusters (Borghese, 2006).
To analyze the function of Stathmin in border cells, stathmin mutants were generated. This was done by imprecise excision of a P element located immediately upstream of the stathmin C transcript. A mutant deleting only the stathmin C isoform (stathminexC), leaving stathmin A and B intact, was homozygous viable and had no effect on border cell migration. A mutant deleting the complete stathmin locus (stathminL27) and four adjacent genes (including Arc-p20, a component of the Arp2/3 complex) was homozygous lethal, and clones of stathminL27 mutant border cells were unable to migrate. Both the lethality and the migration block were rescued by reintroducing ubiquitously expressed stathmin and Arc-p20 at the same time. Reintroducing Arc-p20 alone did not rescue border cell migration, indicating that stathmin is essential for this process. To interfere with stathmin upregulation at the time of migration, a functional stathmin “hairpin”-RNAi construct was expressed in the sensitized stathminexC/stathminL27 background. By using the slbo-GAL4 driver, stathmin RNAi expression could be could specifically targeted to outer border cells right before and during migration. This strongly decreased the amount of Stathmin protein in border cells and caused significant delays in migration. The delays in migration could be reversed by driving higher levels of stathmin expression from a UAS construct. These results identify Stathmin as an important regulator downstream of Slbo. To test whether lack of Stathmin was solely responsible for the slbo phenotype, Stathmin was overexpressed in the slbo mutant background. Migration was not restored, indicating that additional genes downstream of Slbo must also be important (Borghese, 2006).
Singed is an actin-bundling protein related to Fascin, highly expressed in border cells. Fascin is important for the formation of cell protrusions and has been implicated in the control of cell migration, also in vivo. It was confirmed by clonal analysis that Singed protein levels are regulated by Slbo. Despite the strong and regulated expression, migration is normal in border cells mutant for singed. The strongest allele of singed available was used, but it retained a low level of protein expression. In addition, functional overlap may exist between actin regulators. Quail is an actin binding protein of the villin family, and its function in the germline of the ovary genetically overlaps with that of Singed. quail mRNA is also upregulated in border cells relative to follicle cells, and Quail protein is detected in border cells. Quail is structurally similar to Gelsolin, which was also upregulated in border cells, as well as the Gelsolin-related FliI, which was not detectably expressed. However, Gelsolin is enriched in polar cells rather than the migratory outer border cells. As for singed, no migration defects were observed in quail mutant border cells, nor in cells mutant for quail and only one functional copy of singed or vice versa. It was not possible to recover clones of border cells simultaneously mutant for both singed and quail, which is likely to reflect a functional overlap between the two genes at an earlier stage. The simultaneous upregulation of redundant actin regulators may reflect a genetically robust approach to changing the actin cytoskeleton in border cells (Borghese, 2006).
A rather surprising finding of this global expression analysis was that the remaining genes encoding cytoskeleton-associated proteins and upregulated in border cells in a slbo-dependent manner were all “muscle specific”. This included a complete palate of structural genes: muscle actin (57B), muscle myosin heavy chain and light chains, tropomyosin 2 (tm2), troponins, and the calponin-related mp20. The muscle-specific expression has been shown for this group of genes in Drosophila embryos as well as mature muscles. For tropomyosin 2, a GFP gene trap allele was available and, and this allele confirmed expression in border cells as well as in the muscle sheath. The expression profiling indicated that border cells also express the corresponding non-muscle forms such as actin42A, zipper (myosin heavy chain), and sqh (myosin light chain), but at the same level as in follicle cells. The nonmuscle proteins are generally required for many cellular processes, including, where tested, migration of border cells. This raised the question of why this large cluster of muscle-specific structural genes would be turned on in border cells as well. To address this, migration was analyzed of border cells mutant for individual muscle genes for which mutants were available (mhc, mlc2, upheld=troponinT and tm2). Since mhc and mlc2 are essential genes, this was done by clonal analysis. No defects were seen in border cells mutant for mlc2, upheld, or tm2, but clear migration defects were observed in border cells mutant for mhc (mhc1 or mhc3). Thus, while not all of the muscle structural genes are required for border cell migration, at least muscle Mhc expression contributes to effective migration (Borghese, 2006).
Given that both muscle and nonmuscle forms of the same cytoskeletal proteins have a role in border cell migration, their functions are likely to be different. In agreement with this, no genetic interactions were observed between mutants affecting muscle and nonmuscle forms of myosin heavy or light chains. There is precedence for such nonoverlapping functions. For example, Zipper has a unique role in developing muscle cells, which contain plenty of muscle myosin heavy chain. In mammalian cells, different myosin heavy chain isoforms can have distinct subcellular localization. Also, the actin proteins, despite having few amino acid differences, are functionally distinct in vivo (Borghese, 2006).
The muscle gene expression program activated in migratory border cells extended beyond structural genes to regulatory genes. One such gene was bent, encoding a very large titin-like molecule with a myosin light chain kinase domain. Being essential but on the fourth chromosome, bent was not amenable to standard clonal analysis. Genes required for myoblast fusion were also identified, namely, rols/antisocial and rost. mbc, which encodes a DOCK180 family Rac GEF and is required for myoblast fusion, has a role in border cell migration downstream of the PVR guidance receptor. Mbc protein interacts physically with the presumed adaptor protein Rols. Clonal analysis with a strong (likely complete loss-of-function) allele of rols showed defects in border cell migration, suggesting that Mbc and Rols might act together during migration as well. The defect was milder than for mbc, implying that Mbc activity might not be completely dependent on Rols. For the small transmembrane protein Rost, no useful mutants were available. It was also noted that a very closely related and adjacent gene, CG13101, was similarly regulated in border cells and might overlap rost function. Thus, at least mbc and rols function in border cells as well as in muscle (myoblast fusion). Activation of a broad “muscle-specific” gene expression program in border cells may reflect a requirement for a specific subset of the genes within this program (Borghese, 2006).
Previous unbiased genetic approaches to identify genes important for border cell migration have largely identified transcription factors or inducing signals. Changes in cell fate can alter cell behavior dramatically without affecting cell survival, thus still allowing analysis of the mutant cells. The transcription factors themselves often show differential expression. In addition to Slbo, the posttranslationally regulated transcription factor STAT, which is important for border cell migration, was also upregulated in border cells (1.6-fold). The transcription factors that were upregulated in border cells and had mutants available for effects on border cell migration were also tested. aop/yan transcripts were increased 1.9-fold in border cells. In a PiggyBac transposon-based clonal screen for border cell migration defects, an insertion was identified in aop. Complementation analysis confirmed the gene assignment, and quantification of the phenotype showed a clear effect of aop on border cell migration. As expected, border cell migration was strongly affected, but, in addition, clones were rare and morphological abnormalities were seen in other follicle cells as well as in germline cells. Thus, aop may affect the behavior of multiple cell types in the ovary. Another transcription factor, vrille, was also upregulated (over 2-fold). vrille has been implicated in signaling, circadian rhythm, and cellular morphogenesis, but border cells mutant for vrille were largely unaffected and experienced only subtle delays (Borghese, 2006).
The most border cell-enriched RNA encoding a transcription factor, apart from Slbo, was Six4 (4.5-fold). Six4 expression in border cells was confirmed by in situ analysis. Drosophila Six4 is the homeodomain transcription factor most related to mammalian Six4 and Six5. Six family proteins act in complex with proteins of the Eya (Eyes absent) family. eya transcripts were also 2.7-fold enriched in border cells relative to follicle cells of the same stage, and Eya was expressed in a pattern similar to that of six4. Both six4 and eya were expressed in earlier-stage follicle cells as well, and eya has been shown to function at these stages to repress polar cell fate. Follicle cells mutant for six4 expressed a polar cell marker (Fas3) and were functional polar cells, as determined by the ability to induce surrounding anterior follicle cells to become Slbo-positive, migratory border cells. This suggested that Six4 cooperates with Eya in repressing polar cell fate. It had been indicated that Six proteins affect nuclear localization of their Eya partner. The six4 mutant allowed testing this in an in vivo context. Although six4 mutant cells were transformed to functional polar cells, Eya protein was not absent as in the endogenous polar cells, showing that Eya accumulation was independently regulated. However, Eya protein was partially relocalized to the cytoplasm of six4 mutant cells, supporting the hypothesis that Six4 and Eya interact in vivo. Since six4 and eya are both upregulated in outer border cells when they migrate, they are likely to act together in this process as well. However, their earlier roles precludes straightforward loss-of-function analyses in border cells, since “border cell clusters” consisting only of six4 or eya mutant cells are not functional simply because polar cells do not migrate on their own. Overexpression of HA-tagged six4 in border cells interfered with migration, as found for transcription factors required in border cells slbo (Borghese, 2006).
The expression of Six4 in border cells may contribute to activation of the muscle gene program described above. The conserved muscle transcription factor Mef2, an activator of muscle actin and myosin expression, was not detected in border cells by expression profiling or by antibody staining, nor were Twist and Nautilus/MyoD. Six4 is required for development of muscle and other mesodermal tissues in Drosophila. Mutants of C. elegans Unc-39, belonging to the Six4/5 family, also affect muscle/mesodermal differentiation as well as directed cell migration. Mammalian Six5, also called myotonic dystrophy-associated homeodxomain protein (DMAHP), has been analyzed due to its contribution to DM1, and Six4/5 affect normal muscle development. Another transcription factor complex that might contribute to the activation of the muscle program is that of MAL-D (or MRTF) and SRF (serum response factor). The MRTF/SRF complex plays an important role in muscle development in mammals and directly regulates muscle (structural) genes. MAL-D/SRF plays a crucial role in border cell migration and this complex acts to strengthen the cytoskeleton of invasive border cells in response to perceived tension. This mode of regulation makes MAL-D/SRF activity in border cells indirectly dependent on Slbo, which could be responsible for the apparent regulation of the muscle gene cluster by Slbo. The possibility cannot be excluded that Slbo might affect muscle genes directly; the mammalian C/EBP transcription factors are known to regulate different differentiation-specific genes in different contexts (Borghese, 2006).
This study analyzed overall gene expression changes resulting from a transcriptional switch that induces invasive migratory behavior in vivo. The major goal of the analysis was to identify transcriptional changes that directly affect cell behavior and make the cells move. The results indicate that regulation of both the actin cytoskeleton and the microtubule cytoskeleton, likely coordinated regulation, is important for this transition. Identifying Stathmin as an important regulator downstream of Slbo in border cells indicates that microtubule dynamics are critical for border cell migration. Key questions are now how microtubule dynamics affect the process, and whether Stathmin activity is regulated. Two recent findings suggest that Stathmin may be a more general regulator of cell migration: Stathmin-microtubule interactions are spatially regulated in migrating cells in culture, and Stathmin upregulation may promote migration and metastasis of sarcoma cells. The actin cytoskeleton is clearly crucial for cell migration and is controlled by many regulators. The upregulated modulators identified in this study were different from those identified in a whole-genome study of tumor cells selected, in vivo, to be highly motile. There are obviously many differences between these studies; for one, a normal transition to migratory behavior may differ from unrestrained, high motility. The activation of a “muscle-specific” program in migratory border cells was unexpected and provides an intriguing connection between these cells that move and the specialized cells that move an animal (muscle). Overall, the analysis of actin regulators indicates that this is a robust system, with many effectors coregulated, even by one transcription factor. Genetically, this is reflected by minor defects in individual “effector” mutants despite absolute dependence on the transcriptional switch. Further analysis in other systems, and subsequent comparisons, will reveal to what extent the gene expression program employed by border cells to become migratory is a general one (Borghese, 2006).
Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: 4-6 migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. This study reports a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. It was further shown Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and non-motile cells during collective invasion, a phenomenon that may also drive metastatic cancer (Xiang, 2015).
The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (Lin, 2014).
Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. This paper demonstrates a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf–depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis (Matra, 2012).
The Drosophila follicle cell layer encapsulates egg chambers containing 15 nurse cells and one oocyte. The follicle cells comprising this cell layer progress through different developmental stages. During stages 1-5 (S1-5), most follicle cells undergo mitotic divisions, with a few cells exiting the mitotic cycle under Notch activation to form stalk cells separating consecutive egg chambers. During S6-8, all follicle cells exit the mitotic cycle in response to Notch activation and differentiate into an endocycling, polarized epithelium patterned into posterior follicle cells (PFCs), main body cells (MBCs), and anterior follicle cells (AFCs). To examine the effect of inhibiting mitochondrial fission activity in this system, Drosophila follicle cell clones where generated mozygous for a functionally null allele of DRP1 called drp1KG. Clones were identified by lack of a ubiquitin promoter-GFP (UbiGFP) label in their nucleus. The potentiometric dye tetramethylrhodamine ethyl ester (TMRE), which incorporates into the mitochondrial matrix, was used to label mitochondria (Mitra, 2009).
In an S10 egg chamber, nonclonal cells containing a nuclear UbiGFP label have mitochondrial elements widely distributed. Microirradiation at a single point within mitochondria of these cells triggers depolarization (i.e., loss of fluroescent TMRE signal) only at the irradiated site, with little loss of TMRE outside the microirradiated site. This suggested the mitochondrial network of these cells is discontinuous. In drp1KG clones (no UbiGFP label), mitochondria were tightly clustered in a small region of each cell. Single-point microirradiation of mitochondria in a drp1KG clone depolarizes the cell's entire mitochondrial cluster, with complete loss of TMRE signal in 5 s. This indicated that mitochondria in drp1KG clones are highly fused. Reduced mitochondrial fission in drp1KG clones, therefore, causes normally fragmented mitochondrial elements in follicle cells to hyperfuse into a tight cluster (Matra, 2012).
Next, weather presence of drp1KG clones affects follicle epithelial layer organization was examined. In S6-8 egg chambers, follicle cells normally form a single epithelial monolayer. The presence of drp1KG clones, however, disrupts this monolayer arrangement. The effect is most striking in the PFC region, in which drp1KG clones massively overproliferate. The overpopulated clones undergo mitotic cycling even at S10 or later: they incorporate BrdU, demonstrating that they synthesize DNA, and stain with pH3 antibody, indicating that they transit through mitosis. Surrounding heterozygous tissue and drp1KG MBC clones, in contrast, are postmitotic: they neither incorporate BrdU nor stain for pH3. DRP1 depletion thus prevents cell cycle exit primarily in drp1KG PFC clones, leading to their overpopulation in postmitotic egg chambers (Matra, 2012).
As cell cycle exit is a prerequisite for initiating differentiation, whether the drp1KG PFCs are prevented from differentiating was examined. Follicle cells in S6-8 egg chambers normally undergo cell cycle exit to differentiate under the influence of the homeodomain gene Hindsight (Hnt). Notably, clones of drp1KG in the PFC region marked by CD8GFP fail to express Hnt, unlike surrounding nonclonal cells. 95% of drp1KG PFC clones show this phenotype, whereas no drp1KG MBC clonal cells do. Thus, drp1KG PFC clones fail to differentiate (Matra, 2012).
Hnt expression is rescued in all drp1KG PFC clones generated in the background of HA-DRP1 and in 43% of drp1KG PFC clones with DRP1 reintroduced into them. In both conditions, DRP1 expression prevented the clustered mitochondrial phenotype. Lack of differentiation in drp1KG PFC clones, therefore, results from loss of DRP1 activity (Matra, 2012).
Down-regulation of Marf, the Drosophila homologue of mitofusins (Deng, 2008), combined with DRP1 down-regulation in drp1KG PFC clones causes 22% of the clones to now partially express Hnt. Because Marf RNAi expression causes mitochondrial fragmentation when expressed alone or in drp1KG PFC clones, it was concluded that fragmentation of mitochondria reverses the differentiation block in drp1KG PFCs. Therefore, DRP1-driven mitochondrial fission is required for PFCs to differentiate. Loss of function of the inner mitochondrial membrane fusion protein OPA1 caused cell death in this system (Matra, 2012).
Differentiation of Drosophila follicle cells requires Notch receptor activation. Upon ligand binding, the Notch receptor is cleaved to release the Notch intracellular domain (NICD), which redistributes into the nucleus to activate genes required for differentiation. To investigate whether DRP1-driven mitochondrial fission activity acts upstream or downstream of Notch activation in driving PFC differentiation, whether NICD is cleaved and released from the plasma membrane was examined in drp1KG PFC clones. Significant NICD levels are retained on the plasma membrane in drp1KG PFC clones marked by CD8GFP relative to nonclonal cells in S6-8 egg chambers. The Notch extracellular domain (NECD) is also retained on the plasma membrane in these clones, confirming that Notch is inactive. In addition, Cut down-regulation, which occurs in response to Notch activation, does not occur in drp1KG PFC clones. DRP1-driven mitochondrial fission activity thus acts upstream of Notch activation to drive PFC differentiation (Matra, 2012).
NICD loss from the membrane (indicative of Notch activation) increases by 28.2% in drp1KG PFC clones after Marf down-regulation. This suggested that Notch inactivation in drp1KG PFC clones is related to mitochondria being highly fused, with mitochondrial fission a prerequisite for Notch receptor activation in the PFCs. Importantly, expression of an activated Notch (N-Act) domain in drp1KG PFC clones partially overrides the differentiation block in 53% of drp1KG PFC clones, resulting in Hnt expression in these clones. As this occurs without the fused mitochondrial morphology of drp1KG PFC clones changing, the data confirmed that DRP1's role in triggering PFC differentiation is upstream of Notch (Matra, 2012).
Why is DRP1's role in triggering follicle cell differentiation specific to PFCs? Indeed, drp1KG MBC clones show no differentiation block, as Notch activation still occurs in drp1KG MBC clones. Higher levels of bound DRP1 was found in PFCs compared with MBCs after cell permeabilization with digitonin, which may reflect different mitochondrial morphology between PFCs and MBCs. Supporting this, in S6-8 ovarioles it was found that mitochondria in PFCs exist as dispersed fragments both apically and basolaterally, whereas mitochondria in MBCs are tightly clustered at the lateral side of the nucleus. After S9, no observable differences were seen in mitochondrial morphology (Matra, 2012).
Fluorescence loss in photobleaching (FLIP) experiments in follicle cells of S6-8 egg chambers revealed that the dispersed mitochondria of PFCs have less matrix continuity relative to the fused mitochondrial cluster of MBCs. Furthermore, single-point microirradiation caused a 44% loss in TMRE mitochondrial signal per MBC compared with a 12% loss per PFC. The rapid loss of mitochondrial TMRE signal in MBCs was similar to drp1KG clonal cells, with mitochondrial morphology in wild-type MBCs indistinguishable from that of drp1KG MBC clones. Together, the observed differences in mitochondrial organization and bound DRP1 levels in PFCs and MBCs suggested greater DRP1-driven mitochondrial fission activity occurs in PFCs relative to MBCs. This corroborates findings that PFCs, unlike MBCs, differentiate under the influence of DRP1 (Matra, 2012).
PFCs are known to be specified by EGF receptor (EGFR) signaling. In egfrt1/egfrt1 egg chambers (hypomorphic allele of EGFR), mitochondria in PFCs are primarily clustered to one side of the nucleus, in contrast to those in wild-type or egfrt1/+ egg chambers, in which mitochondria are dispersed throughout cells. A similar clustering of mitochondria occurs when a dominant-negative (DN) form of EGFR (EGFR-DN) is clonally expressed in the PFC population. Because PFC mitochondria cluster/fuse in the absence of EGFR signaling, the data suggest that EGFR activation in PFCs promotes mitochondria fragmentation in these cells. This could explain why MBCs, which do not receive the EGFR signal, have fused mitochondria. The underlying basis for how EGFR signaling influences mitochondrial dynamics (by altering fission or fusion components) requires further investigation (Matra, 2012).
Interestingly, PFCs expressing EGFR-DN did not escape differentiation in spite of having clustered mitochondria. This may imply that a highly fused mitochondrial cluster may only allow escape from differentiation in the context of activated EGFR signaling. Indeed, EGFR-DN expression in drp1KG PFC clones (with fused mitochondria) partially induces differentiation (i.e., Hnt expression) in 40% of the clonal cells compared with no Hnt expression in drp1KG PFC clone. Expression of an activated form of EGFR (EGFR-Act) did not induce differentiation in drp1KG PFC clones. This explains why MBCs, which are not exposed to the EGFR ligand, do not proliferate under DRP1 down-regulation. Thus, cross talk exists between mitochondria and the EGFR signaling pathway in postmitotic PFCs, which helps cells decide whether to differentiate or continue in the mitotic cycle (Matra, 2012).
Whether DRP1 activity is important for regulation of cell cycle exit of mitotic follicle cells to allow onset of differentiation was investigated. The majority of follicle cells in S1-5 (during which all cells are mitotic) have fragmented mitochondria, suggesting that DRP1-dependent fission activity is high. drp1KG follicle cell clones introduced into the mitotic follicle cell layer and lacking UbiGFP harbor characteristic mitochondrial clusters. Clones also contain more pH3-positive cells and have qualitatively greater incorporation of BrdU relative to nonclonal tissue, with Cut expression unaltered. Without DRP1, therefore, S1-5 follicle cells undergo faster mitotic cycling (Matra, 2012).
To test whether DRP1 activity is necessary for mitotic cells to differentiate, Marf RNAi was expressed to allow unopposed DRP1 activity in S1-5 egg chambers. Strikingly, Marf RNAi expressing follicle cell clones (marked by CD8GFP) show premature expression of Hnt, whereas neighboring nonclonal mitotic follicle cells do not. The effect is not restricted to any stage or region of the mitotic follicle cell layer. The Marf RNAi follicle cell clones exhibit increased mitochondrial mass as assessed by HSP-60 staining and MitoTracker loading, similar to that reported previously from mitofusin knockout mice . Importantly, drp1KG follicle cell clones expressing Marf RNAi do not show premature differentiation; Hnt and HSP-60 expression levels are comparable with wild-type cells. Therefore, the premature differentiation of Marf RNAi clones is dependent on DRP1. This indicates that DRP1-driven mitochondrial fission activity is required for mitotic follicle cells to exit the cell cycle and initiate their differentiation regimen (Matra, 2012).
Because of DRP1's role in differentiation, lack of DRP1 should generate developmental defects. Consistently, DRP1 down-regulation in early follicle cells in the germarium inhibits stalk cell formation, required to separate consecutive egg chambers. The missing stalk cells in egg chambers, encapsulated by early drp1KG follicle cell clones, leads to fused egg chambers containing pH3-labeled drp1KG clonal cells that lack UbiGFP. FasIII-enriched polar cells, known to induce stalk cells, are seen in wild-type ovarioles but are absent in the drp1KG clonal follicle cell population. Lack of polar cells is not the basis of cell proliferation of drp1KG PFCs because FasIII-positive polar cells appear in the surrounding heterozygous tissue. In addition, compound egg chambers with drp1KG follicle stem cell clones frequently arise, including egg chambers with 30 nurse cells and two oocytes (Matra, 2012).
Down-regulation of DRP1 also causes developmental defects in the postmitotic follicle cell layer. There, in 22% of the cases, drp1KG PFC clones fail to trigger migration of the oocyte nucleus toward the anterior. The postmitotic stage drp1KG phenotypes resemble loss of function of the Hippo-Salvador-Warts pathway, which has tumor suppressor effects in higher organisms, including mice (Matra, 2012).
The observed link between cell differentiation and mitochondrial fission state during oogenesis could relate to cyclin E, which controls S-phase entry. Indeed, inhibition of mitochondrial ATP synthesis in a cytochrome oxidase mutant promotes specific degradation of cyclin E (but not other cyclins) and blocks S-phase entry in Drosophila. In fibroblasts, cyclin E levels increase under conditions of DRP1 inhibition. In Drosophila follicle cells cyclin E levels were found to increase when DRP1 is down-regulated and decrease when Marf is down-regulated. This suggests that DRP1-driven mitochondrial fission activity may cause cell cycle exit by lowering cyclin E levels to allow differentiation (Matra, 2012).
The results support a model in which mitochondrial fission/fusion dynamics regulates cell differentiation across the follicle cell layer of the Drosophila ovariole (see A model for mitochondria's role in cell fate determination). In mitotic stages, increased DRP1-driven mitochondrial fission is required for cell cycle exit as noted in premature DRP1-dependent differentiation of Marf RNAi clones and enhanced proliferation of drp1KG clones. During postmitotic transition, activation of EGFR in the posterior region causes mitochondrial fragmentation. This, in turn, permits cell cycle exit and Notch activation, which drives PFC differentiation. In drp1KG PFC clones with fused mitochondria, therefore, Notch remains inactive, and cells proliferate. In the main body region, not exposed to the EGFR ligand, postmitotic differentiation and patterning occur in the absence of DRP1. Thus, cell proliferation/differentiation mechanisms have an intimate relationship to mitochondrial morphology and function during follicle layer development (Matra, 2012).
Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. This study reports that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).
This study reveals that Lpd colocalizes with the Scar/WAVE complex at the very edge of lamellipodia and directly interacts with this complex by binding to the Abi-SH3 domain. Active Rac directly binds Lpd, thereby regulating the interaction between Lpd and the Scar/WAVE complex. It is therefore postulated that Lpd acts as a platform to link active Rac and the Scar/WAVE complex at the leading edge of cells to regulate Scar/WAVE-Arp2/3 activity and thereby lamellipodium formation and cell migration (Law, 2013).
Knockdown of Lpd expression or KO of Lpd highly impaired lamellipodium formation, phenocopying the effect of Scar/WAVE complex knockdown on lamellipodium formation. Conversely, it was observed that overexpression of Lpd increased lamellipodia size in Xenopus NC cells, and this was dependent on the interaction with Abi, linking it to the Scar/WAVE complex. Overexpression of Pico, the Lpd fly orthologue, aberrantly increased the number and frequency of cellular protrusions at the rear of border cell clusters in a Scar-dependent manner, which suggests that the regulation of Scar/WAVE by Lpd is evolutionary conserved. Collectively, these data suggest that Lpd functions to generate lamellipodia via the Scar/WAVE complex (Law, 2013).
Lpd or Pico knockdown or Lpd KO impaired cell migration in vitro and in vivo in Drosophila, Xenopus, and mice. Lpd KO or knockdown cells were unable to migrate via lamellipodia but instead migrated very slowly by extending filopodia. The same residual migration mode had been observed for Arp2/3 knockdown cells. Arp2/3 is activated by the Scar/WAVE complex to regulate cell migration. It was also observed that both Lpd and Abi knockdown impaired NC migration in vivo. Consistently, it was found that Lpd and Abi-Scar/WAVE are in the same pathway regulating cell migration. This is consistent with recent studies suggesting that the Lpd orthologue in C. elegans, mig-10, genetically interacts with abi-1 to regulate axon guidance, synaptic vesicle clustering, and excretory canal outgrowth in C. elegans (Stavoe, 2012; Xu, 2012; McShea, 2013). Collectively, these results suggest that Lpd functions in cell migration via the Scar/WAVE complex in mammalian cells, Xenopus NC cells, and Drosophila border cells (Law, 2013).
Lpd not only interacts with the Scar/WAVE complex but also directly binds to Ena/VASP proteins. Ena/VASP proteins regulate actin filament length by temporarily preventing capping of barbed ends and by recruiting profilin-actin to the growing end of actin filaments. In contrast, the Scar/WAVE-Arp2/3 complexes increase branching of actin filaments. Lamellipodia with a highly branched actin network protrude more slowly but are more persistent, whereas lamellipodia with longer, less branched actin filaments protrude faster but are less stable and quickly turn into ruffles. It was observed that Lpd overexpression increases cell migration in a Scar/WAVE- and not Ena/VASP-dependent manner. This is consistent with a predominant function of Scar/WAVE downstream of Lpd to regulate a highly branched actin network supporting persistent lamellipodia protrusion and cell migration. Other actin-dependent cell protrusions such as axon extension or dorsal ruffles of fibroblasts require Lpd-Ena/VASP-mediated F-actin structures (Law, 2013).
Collective cell migration describes a group of cells that moves together and affect each other, and various types of collective cell migration exists during development and cancer invasion. Xenopus NC cells migrate as loose streams, whereas Drosophila border cells migrate as a cluster of cells with close cell-cell contacts. This study found that Rac regulates Lpd and Scar/WAVE interaction and that both are required for Xenopus NC migration, which is consistent with previous work in which Rac activity mediates this type of migration. Similarly, NC-derived melanoblast migration in the mouse depends on Rac-Scar/WAVE-Arp2/3, and it was found that Lpd functions in this process as well (Law, 2013).
Drosophila border cell clusters migrate through the fly egg chamber in two phases: an early part characterized by large and persistent front extensions, which are regulated predominantly by PVR (the fly PDGF receptor); and a late part characterized by dynamic collective 'tumbling' behavior. Surprisingly, Pico overexpression resulted in the appearance of a higher proportion of rear facing extensions, a phenotype previously observed with dominant-negative PVR, causing premature tumbling of the border cell cluster. This suggests that Pico function is normally tightly controlled to stabilize specific extensions and functions also in guidance of collective cell migration. Because Lpd-Scar/WAVE control single cell migration as well as collective cell migration, this suggests that they function as general regulators of cell migration (Law, 2013).
Collectively, this study has identified a novel pathway in which Lpd functions as an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).
Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochrome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals (Tootle, 2011).
These studies show that a large fraction of the genes involved in eggshell production can be identified by simply scoring for stage-specific changes in transcript levels during late oogenesis. Not only were virtually all of the previously known structural genes involved in producing yolk, the vitelline membrane and the chorion identified, but we also discovered at least 19 new candidate eggshell proteins. Despite the simplicity of the protocol, the expression profiles revealed by these experiments agreed closely with previous studies, including the temporal programs reported by Fakhouri (2006) for 10 minor chorion proteins as determined by whole mount in situ hybridization. Many of the genes identified as eggshell genes are included among 81 genes reported as candidate targets of Egfr signaling by Yakoby (2008). Additionally, the expression of many genes involved in follicle maturation are spatially regulated within the folliclar epithelium, and the method used in this study could serve as an efficient pre-screen before undertaking such studies (Tootle, 2011).
It is interesting to compare these results with studies of eggshell proteins isolated from whole ovaries and analyzed by mass spectrometry (Fakhouri, 2006). The current analyses confirm many of the 22 genes identified in that study, including CG11381 (FBgn0029568), CG13083 (FBgn0032789), CG13084, CG13114, CG14796 (FBgn0025390), CG15570, CG4009, and CG31928. CG3074 (FBgn0034709) and CG13992 (FBgn0031756) are expressed at lower levels during stage 10 and may encode vitelline membrane proteins. The value of using temporal regulation as a criterion to identify eggshell components is highlighted by the fact that transcripts corresponding to the minor basement membrane components identified by Fakhouri (2006) remain constant during eggshell formation, suggesting that they derive from matrix material adherent to the purified eggshells, rather than representing true eggshell constituents (Tootle, 2011).
Like the known eggshell genes, many of the 19 new candidate eggshell genes reside within existing or new gene clusters. For example, within the vitelline membrane gene cluster at 26A, it is predicted that CG13998 encodes a novel Vm protein. CG13998 is expressed with the same stage specificity as other Vm proteins encoded within the 26A cluster, but is much less abundant. It may encode a rare or spatially restricted component of the vitelline membrane (Tootle, 2011).
A number of the putative eggshell genes encode for proteins with mucin-like domains (CG32774/Muc4B, CG32642/Mur11D, CG32602/Muc12Ea). All three were scored as ovary-specific in FlyAtlas. Mucin domains are heavily glycosylated and proteins with such domains often create a gel-like secretion. Mucins are known to function as components of chicken eggshells and coat the reproductive tract in some animals. CG32774/Muc4B is expressed mainly at S12. Based on the functions of mucin proteins and the timing of gene expression, it is postulated the Muc4B may be a component of the wax layer that is located between the vitelline membrane and the chorion. The other mucin-like domain proteins are expressed at S14 and may mediate chorion hardening, protection against infection, or serve as a coating necessary for passage through the oviduct (Tootle, 2011).
Another class of predicted eggshell genes encode for proteases and protease inhibitors. Such proteins have recently been shown to be a major class of chicken eggshell components. CG31928, CG31926, and CG31661 are aspartic proteases. Proteases may act to process other chorion proteins into their mature form, and/or contribute to eggshell hardening. Previously, the protease CG31928 has been shown by in situ hybridization to exhibit a posterior restriction. This raises the idea that proteases may be spatially restricted to alter chorion structure at specific regions that are important for subsequent function. It seems likely that other proteases may be restricted anteriorly, to the operculum area, to alter the eggshell structure to subsequently mediate larval hatching. In addition to proteases, it was found that a number of protease inhibitors are eggshell candidates, including CG15721 (S14), CG12716 (S14), CG1077 (S12), and CG15418 (S12; FBgn0031554). Stage 12-expressed protease inhibitors may regulate eggshell proteases, while stage 14-expressed inhibitors may perform anti-microbial activities. Both the proteases and their inhibitors may also contribute to the embryo's ability to reutilize eggshell components for its development (Tootle, 2011).
The nature of the peroxidase that crosslinks the eggshell has been controversial. Various proteins have been suggested to function as the crosslinking peroxidase, including Pxd (FBgn0004577) and Pxt. This study found pxt, the COX-like enzyme, is expressed and pxd is absent throughout all stages of egg maturation. In contrast, the eggshell protein and putative peroxidase CG4009 is very highly expressed during S12. It is proposed that CG4009 is the peroxidase that crosslinks the eggshell (Tootle, 2011).
In addition to known and novel eggshell components, many temporally regulated genes expressed during late follicle development were identified. A number of putative lipid-processing genes exhibit stage specific expression, suggesting a role in yolk or pheromone production. CG9747 (FBgn0039754) encodes for an acyl-CoA Δ11-desaturase, which is likely to desaturate palmitate; such enzymes are important for pheromone biosynthesis in other insects. CG8303 (FBgn0034143) is highly expressed as stages 9/10A and 10B, and encodes for an acyl-CoA reductase, which activates fatty acids by adding CoA. Additionally two Elvol (elongation of very long chaing fatty acids) encoding genes, bond (CG6921; FBgn0260942) and CG2781 (FBgn0260942), are expressed at S10A and S10B, respectively. Bond has previously been shown to be required for both male and female fertility, and by in situ hybridization appears to be expressed during oogenesis in the main body follicle cells at stages 9 and 10. These genes may contribution in a manner that is not currently understood to mediate the production of lipid yolk, or they may function in the production of lipid-based signals that contribute to egg maturation (Tootle, 2011).
Pxt mutations partially uncouple morphological development and gene expression. Yolk protein genes turn off normally in pxt mutant follicles, but vitelline membrane genes continue to be expressed longer than normal. Some chorion genes turn on earlier than normal, while the expression of others is delayed or prolonged. Many possible mechanisms may underlie these changes. However, the possibility that Pxt coordinates the production of Prostaglandins (PGs) that interact with other mechanisms to precisely control egg maturation is particularly interesting (Tootle, 2011).
In all sexually reproducing organisms the growth and development of the somatic and germ cells are mutually dependent and must be coordinated. Such coordination requires bi-directional communication. Historically, somatic cells were thought to regulate follicle development, including maintaining meiotic arrest, promoting meiotic resumption, and suppressing oocyte transcription prior to nuclear maturation. It has more recently been shown that the oocyte also signals to the soma. Oocyte signaling is necessary for follicular formation, and regulating the proliferation and differentiation of the somatic cells. It is generally thought that the oocyte has a greater influence on the soma early in follicular development and this is reversed during the later stages (Tootle, 2011).
There is emerging evidence that PG signaling coordinates germline and somatic development within mammalian follicles. While both oocyte and somatic maturation are delayed in COX2 knockout mice, it has been shown that the PGs are required in the soma for fertility. Specifically, COX2 is required in the somatic cells for cumulus (somatic) cell expansion and survival. However, meiotic resumption is not controlled by PGs from the soma. These germline and somatic events must be coordinated for the follicle to be competent for fertilization. This study found that PG signaling is required for both germline and somatic development during Drosophila follicle development. Fertility requires both of these signals. Specifically, PG signaling within the germline is necessary for mediating nurse cell dumping, the contractile process by which the oocyte is supplied with materials required for embryonic development, while PG signaling within the follicle cells is needed to regulate the timing of eggshell gene expression and subsequent eggshell structure. Thus PG signals, from insects to mammals, maintain the synchronized development of the germline and somatic cells within the individual follicle (Tootle, 2011).
Female reproduction is regulated by a complement of hormones that are cyclically produced and secreted. One such hormone that interacts with PG signaling in mammals is oxytocin. Oxytocin plays critical roles in regulating the function of the corpus luteum, a transient endocrine organ that secretes hormones to regulate the menstrual cycle and the early stages of pregnancy. In the absence of pregnancy, PGF2alpha stimulates the release of oxytocin to mediate luteolysis or the regression of the corpus luteum. During parturition, oxytocin and PGF2alpha also play critical roles. Oxytocin initiates labor, inducing PGF2alpha, which maintains labor and dilates the cervix (Tootle, 2011).
PGs and estrogen co-regulate each other in multiple cells types, including breast cancer cells. Breast tissue is the largest producer of estrogen in post-menopausal women; aromatase, Cyp19, leads to the production of estradiol. There is a high correlation between aromatase and COX2 expression in human breast cancer samples. Specifically, PGE2 signals via cAMP and PKA to stimulate a promoter upstream of cyp19, leading to increased aromatase expression. Autocrine and paracrine feedback loops via estradiol subsequently increase PGE2 secretion. Therefore, in breast cancer cells, PG and estrogen signaling are intimately linked (Tootle, 2011).
PGs and estrogen also interact in endometriotic tissue. Both PGE2 and PGF2alpha are excessively produced in uterine and endometriotic tissues of women with endometriosis. In the endometriotic stromal cells, PGE2 stimulates the expression of all the steroidogenic genes needed to synthesis estradiol from cholesterol. This occurs via PGE2 activation of cAMP/PKA signaling which upregulates of the expression of steroidogenic acute regulatory gene (StAR) and cyp19 . The expression of these steroidogenic genes is regulated by Steroidogenic Factor 1 (SF1), a nuclear hormone receptor. PGE2 signaling leads to SF1 out competing other transcription factors, Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) and Wilms' tumor-1 (WT-1), for binding to steroidogenic gene promoters. Thus, PG signaling coordinates the expression of all steroidogenic genes (Tootle, 2011).
These results encourage future efforts to further establish the roles for PG signaling during Drosophila egg maturation and specifically, to learn how PGs are connected to steroid hormones. The Drosophila hormone ecdysone plays several critical roles during oogenesis. The loss of ecdysone signaling arrests follicle development at stage 8. Additionally, ecdysone signaling is needed to control the onset of chorion gene amplification, and to activate eggshell gene expression via transcriptional regulation. Temporally programmed changes in ecdysone levels may contribute to the timed control of eggshell gene expression. These studies provide a foundation for further dissecting the roles of Pxt and ecdysone-mediated signaling during late follicle development. If important aspects of these interactions have been conserved during evolution, the Drosophila ovary may emerge as a model for understanding the cellular and molecular changes underlying mammalian follicular maturation, endometriosis and infertility (Tootle, 2011).
Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains over 7,000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. The 84 genes known to be expressed during D. melanogaster oogenesis were cross-listed with the ~1200 listed genes of the FlyLight collection, and 22 common genes were found that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ~3kb in length, the vast majority of fragments show one type of a spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, a significant enrichment of CRMs was found in the first intron of the associated genes' model. In addition, the use was demonstrated of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. This screen provides further evidence that complex gene-patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains (Revaitis, 2017).
Morphogenesis of the respiratory appendages on eggshells of Drosophila species provides a powerful experimental system for studying how cell sheets give rise to complex three-dimensional structures. In Drosophila, each of the two tubular eggshell appendages is derived from a primordium comprising two distinct cell types. Using live imaging and three-dimensional image reconstruction, it was demonstrated that the transformation of this two-dimensional primordium into a tube involves out-of-plane bending followed by a sequence of spatially ordered cell intercalations. These morphological transformations correlate with the appearance of complementary distributions of myosin and Bazooka in the primordium. These distributions suggest that a two-dimensional pattern of line tensions along cell-cell edges on the apical side of the epithelium is sufficient to produce the observed changes in morphology. Computational modeling shows that this mechanism could explain the main features of tissue deformation and cell rearrangements observed during three-dimensional morphogenesis (Osterfield, 2013).
The formation of 3D structures from epithelial sheets is a key feature of embryonic development. The Drosophila egg chamber provides a powerful model for studying these processes. This study analyzed how the dorsal appendage tubes emerge from the follicular epithelium. It was found that tube formation in this system preserves the integrity of the follicular epithelium and proceeds through a combination of sheet bending and lateral cell rearrangements. Based on the localization patterns of myosin and Baz, it is hypothesized that these events are caused by forces within the apical surface of the sheet. The special feature of this model is that it results in tissue transformations similar to those observed experimentally utilizing tensions generated exclusively in the 2D apical surface. Note that previous 3D extensions to the vertex model have modeled cells as 3D prisms. In contrast, the current approach involves allowing an essentially 2D object, the apical surface of the epithelial sheet, to move and deform in 3D space. The morphological changes in this model are driven by apical processes, without consideration of other cellular features such as volume constraints and active processes on the basal surface. At this point, the feasibility of this model is supported mainly by the computational studies that demonstrate how a pattern of tensions within a sheet can first bend the sheet and then initiate ordered intercalations, forming the seam of the tube. The patterns of apical tension predicted by this model agree qualitatively with the localization patterns of myosin in the appendage primordium at different stages of tube formation. In the future, however, this model should be tested by direct measurements of tensions, for example by laser ablation, and extended to account for processes on the basolateral cell surfaces as well as the processes associated with tube elongation (Osterfield, 2013).
Several mathematical models have been proposed for bending of cell sheets. One mechanism, working in both plants and animals, relies on spatial differences in cell proliferation, which causes tissue deformations. Since the follicle cells do not divide during the stages analyzed in this work, this mechanism does not apply to dorsal appendage morphogenesis. Other mechanisms, such as those put forward for vertebrate neurulation and ventral furrow formation in Drosophila, work through apical constriction, which occurs in the current system as well. However, one additional element common to these models is that bending is generated by a difference in apical versus basal properties. This clearly does not drive dynamics in the current model, which considers only the apical surfaces. Instead, out-of-plane displacements of the appendage primordium can be understood as a manifestation of buckling, whereby mechanical forces within the sheet give rise to states that can be either flat or bent, with the bent state having a lower energy. It will be interesting to explore whether similar models can predict out-of-plane deformations in other systems, such as those seen during eversion of imaginal discs (Osterfield, 2013).
In the current model, patterned apical tension is sufficient to explain not only buckling but also ordered intercalation. Although cell intercalation in the simulations is spatially ordered in a manner reminiscent to that seen in live imaging, there are some differences that should be interesting to explore in the future. In the imaging data, the floor cells eventually form two rows of floor cells separated by a relatively straight seam, while in the simulations the seam is more uneven and sometimes disrupted by the presence of one or more floor cells between these row. This suggests the possible existence of additional mechanisms for highly ordered intercalation, beyond those included in the model. One potential mechanism to explore further both experimentally and computationally is the possible formation of rosettes, since recent studies in other systems indicate that the use of rosettes in addition to T1 transitions may increase the efficiency of intercalation-mediated processes such as migration and tissue elongation (Osterfield, 2013).
In the current model of dorsal appendage formation, patterned line tension plays a key role. Future work will be needed to address the molecular mechanisms by which patterns of tension are established. Included among the genes with patterned expression in the late follicular epithelium are several that encode proteins involved in cytoskeleton regulation or cell-cell adhesion. Mutations in some of these genes result in dorsal appendage defects, but whether these genes work through regulating tension or through some other process has been largely unexplored (Osterfield, 2013).
Tube formation is a common outcome of epithelial morphogenesis. Sealing or closure of the tube is one of the least understood aspects in systems where tubes form by wrapping, as in the vertebrate neural tube or the Drosophila ventral furrow. The dorsal appendage tube appears to be sealed by spatially ordered lateral cell rearrangements. This suggests that lateral rearrangements may play a role in seam sealing in other cases of wrapping as well. Lateral rearrangements alone cannot be sufficient to drive morphogenesis in cases where the tube becomes discontinuous from its parental sheet, but future studies may reveal whether lateral rearrangements nevertheless play a key role in such systems (Osterfield, 2013).
Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. This study demonstrates that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells. Escort cells produce both Wingless and Hedgehog ligands for the FSC lineage, and Wingless signaling is specific for the FSC niche whereas Hedgehog signaling is active in both FSCs and daughter cells. In addition, this study shows that multiple escort cells simultaneously encapsulate germ cell cysts and contact FSCs. Thus, FSCs are maintained in a dynamic niche by a non-dedicated population of niche cells (Sahai-Hernandez, 2013).
Taken together, the results of this study challenge the notion that the
FSC niche is maintained by gradients of ligands produced solely at
distant sites. Instead, the data indicate that the FSC
niche has a more canonical architecture in which at least some key
niche signals are produced locally, although the FSC niche might also differ
from other well-characterized niches in some ways, such as the extent
to which it remodels during adulthood. Notably, the results do not
contradict the observation that Hh protein relocalizes from apical cells
to the FSC niche during changes from a poor to a rich diet, as the flies were consistently maintained on nutrient-rich
media. It will be interesting to investigate how such distantly produced
ligands interact with locally produced niche signals to control FSC
behavior during normal homeostasis and in response to stresses (Sahai-Hernandez, 2013).
In addition, the results confirm and extend the conclusion that Wg
acts specifically on FSCs and ISCs, thus highlighting the role of Wg as a specific epithelial stem
cell niche factor. As in other types of stem cell niches, this specificity
could be achieved through multiple mechanisms, including local
delivery of the Wg ligand to the niche and crosstalk with other
pathways such as Notch and Hh, which are known to interact with the
Wg pathway. Although the precise function(s) of Wg
signaling in FSCs is unclear, the observation that a reduction in Wg
ligand results in a backup of cysts near the FSC niche at the region
2a/2b border and fused cysts downstream from the FSC niche
suggests that one role is to promote FSC proliferation. In addition, the
finding that FSC daughter cells with ectopic Wg signaling fail to form
into a polarized follicle epithelium suggests that Wg signaling might also promote self-renewal in
FSCs by suppressing the follicle cell differentiation program.
By contrast, observations and published studies indicate that
Hh signaling is not specific for the FSC niche but instead constitutes
a more general signal that derives from multiple sources and
regulates proliferation and differentiation in both FSCs and
prefollicle cells. Consistent with this conclusion, Hh signaling is
active throughout the germarium and
is required both in FSCs to promote self-renewal and in prefollicle cells
to promote development toward the stalk and polar lineages (Sahai-Hernandez, 2013).
Finally, multicolor labeling of somatic cells in the
germarium indicated that multiple densely packed escort cell
membranes surround region 2a cysts and contact the FSC niche.
Although the possibility cannot be ruled out that one or more cells
in this region are dedicated FSC niche cells, observations
strongly suggest that at least some escort cells contribute to both
germ cell development and the FSC niche. Since these escort cells
are dynamic, constantly changing
their shape and position to facilitate the passage of germ cell cysts,
it is perhaps somewhat surprising that the FSCs are so stable in the
tissue. Indeed, the rate of FSC turnover is comparable to that of
female GSCs, which are maintained by a dedicated and more static
niche cell population. It will be
interesting to investigate how this dynamic population of escort
cells is able to maintain such a stable microenvironment for the
FSCs. One possibility is that redundant sources of niche signals
may allow niches of this type to partially break down and reform
as needed to rapidly accommodate the changing demands of the tissue (Sahai-Hernandez, 2013).
The current observations reinforce several themes that are emerging from
recent studies of stem cell niches in different epithelial tissues. First,
as in the FSC niche, the Wnt/Wg signaling pathway is a key stem
cell niche signal in many Drosophila and mammalian epithelial
tissues. Second, in several epithelial tissues, the stem cell self-renewal
signals are also known to be produced by differentiated
cells rather than a dedicated niche cell population. For example,
Drosophila ISCs of the gut receive self-renewal signals from both nearby
enterocytes and the surrounding visceral muscle. Likewise, mammalian ISCs at the
base of the crypt receive self-renewal signals from Paneth cells, which are adjacent secretory cells with antimicrobial
functions. Lastly, several epithelial niches have recently been shown
to have a transitory capacity that may resemble the dynamic nature
of the FSC niche. For example, stem cell niches can form de novo
in the Drosophila intestine to accommodate increased food
availability, and in the mammalian skin in
response to hyperactive Wnt signaling. In
addition, mammalian ISCs produce niche cells in vivo and can spontaneously reform a niche in culture. In all of these examples, it seems likely that the relationship
between the epithelial stem cell and its niche is not static, but instead
flexible and dynamic. Further studies of the Drosophila FSC niche
and these other experimental models will continue to provide
insights into the mechanism by which a dynamic epithelial stem cell
niche functions (Sahai-Hernandez, 2013).
Addressing the complexity of organogenesis at a system-wide level requires a complete understanding of adult cell types, their origin, and precursor relationships. The Drosophila ovary has been a model to study how coordinated stem cell units, germline, and somatic follicle stem cells maintain and renew an organ. However, lack of cell type-specific tools have limited the ability to study the origin of individual cell types and stem cell units. This study used a single-cell RNA sequencing approach to uncover all known cell types of the developing ovary, reveal transcriptional signatures, and identify cell type-specific markers for lineage tracing. This study identifies a novel cell type corresponding to the elusive follicle stem cell precursors and predicts subtypes of known cell types. Altogether, this study reveals a previously unanticipated complexity of the developing ovary and provide a comprehensive resource for the systematic analysis of ovary morphogenesis (Slaidina, 2020).
This study has identified cell type-specific marker genes, which now open the targeted use of hundreds of publicly available GFP fusion constructs that can be used for cell labeling, live imaging, and functional studies. For example, a GFP fusion of a highly specific Sheath cell (SH) marker drm (see scRNA-seq experiment design and statistics), can be used to label SH, and additional lines exist for other cell types. This study used a number of publicly available Gal4 drivers for lineage analyses. While some of these were expressed broader than expected from mRNA expression patterns, it was possible to identify Gal4 driver lines for lineage tracing of each larval ovary cell type. In particular, these lines helped determine the adult descendants of the swarm cells and identified the long sought-after follicle stem cell progenitors. Going forward, the cell type-specific markers identified in this study can be used for further tool building to more specifically and completely target individual cell types. For example, strategies involving Gal80TS and split-Gal4 systems may improve driver specificity and avoid expression in other tissues. GLAD analysis grouped cell type signature genes according to their molecular and cellular functions. Predicted cellular functions and protein classes enriched in each cell type will provide new insight into how cells in the developing ovary interact, how stem cell units are established, and how these precursor cell interactions support the morphogenesis and homeostasis of the adult ovary (Slaidina, 2020).
A major finding of this study is the identification of a follicle stem cell and follicle cell progenitor population. The results show that the transcriptional signatures of precursors for the adult FSC (FSCPs) and intermingled cells (ICs) are similar. This could indicate that these two cell types are specified from a common progenitor. In support, the FSCP marker gene bond is detected as early as EL3 in a broad expression domain spanning both the FSCP and IC progenitors. bond may be expressed in the common progenitor pool and later become restricted to the FSCPs, or the bond-expressing FSCPs may be initially dispersed and later migrate posteriorly. In addition to common developmental origins, an overlap in transcriptional signatures may also reflect shared functions. Consistently, ICs and FCs both intimately interact with germ cells and guide their differentiation; thus, analyzing the overlap between the IC and FSCP transcriptional signatures might reveal the nature of IC/FSCP to GC signaling, and shed light on stem cell-to-support cell communication in general (Slaidina, 2020).
Altogether, this study provides a systems-wide overview of cell types, and their transcriptional profiles and signatures in the developing Drosophila ovary. This resource will facilitate future studies, leading to a better understanding of how stem cell populations are specified, regulated, and maintained in the context of a growing organ, and more general, how a complex interplay of several cell types achieves to build an organ. Future scRNA-seq experiments using additional stages of development (earlier larval, pupal, adult) or using scRNA-seq methods that allow simultaneous lineage tracing, like scGESTALT will allow identification of the complete lineage relationships between the ovarian cell types. Moreover, perturbing functions of individual cell types will provide information about cellular processes that are coordinated between the cells and how this coordination is achieved. Together, this work should provide an invaluable resource for the stem cell and developmental biology research communities (Slaidina, 2020).
The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. This study reports the first genome-scale in vivo RNAi screen for genes controlling epithelial development. By directly analysing cell and tissue architecture, 1125 genes were identified that were assigned to seven different functions in epithelial formation and homeostasis. The significance of the screen was validated by generating mutants for Vps60, a component of the ESCRT machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. The data distinguish two new domains with differential proliferation control outside of the stem cell niche. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT mediated proliferation control. Adjacently, another domain is defined by loss of ESCRT impact on cell division. Thus, during development epithelial cells pass through different modes of proliferation control. The switch between these modes might reflect regressing stemness of epithelial cells over time (Berns, 2014).
Stem cell niches provide localized signaling molecules to promote stem cell fate and to suppress differentiation. The Drosophila melanogaster ovarian niche is established by several types of stromal cells, including terminal filament cells, cap cells, and escort cells (ECs). This study shows that, in addition to its well-known function as a niche factor expressed in cap cells, the Drosophila transforming growth factor β molecule Decapentaplegic (Dpp) is expressed at a low level in ECs to maintain a pool of partially differentiated germline cells that may dedifferentiate to replenish germline stem cells upon their depletion under normal and stress conditions. This study further reveals that the Dpp level in ECs is modulated by Hedgehog (Hh) ligands, which originate from both cap cells and ECs. Hh signaling exerts its function by suppressing Janus kinase/signal transducer activity, which promotes Dpp expression in ECs. Collectively, these data suggest a complex interplay of niche-associated signals that controls the development of a stem cell lineage (Liu, 2015).
Animals from flies to humans adjust their development in response to environmental conditions through a series of developmental checkpoints, which alter the sensitivity of organs to environmental perturbation. Despite their importance, little is known about the molecular mechanisms through which this change in sensitivity occurs. This study has identified two phases of sensitivity to larval nutrition that contribute to plasticity in ovariole number, an important determinant of fecundity, in Drosophila melanogaster. These two phases of sensitivity are separated by the developmental checkpoint called critical weight; poor nutrition has greater effects on ovariole number in larvae before critical weight than afterwards. This switch in sensitivity results from distinct developmental processes. In pre-critical weight larvae, poor nutrition delays the onset of terminal filament cell differentiation, the starting point for ovariole development, and strongly suppresses the rate of terminal filament addition and the rate of increase in ovary volume. Conversely, in post-critical weight larvae, poor nutrition only affects the rate of increase in ovary volume. These results further indicate that two hormonal pathways, the insulin/insulin-like growth factor and the ecdysone signalling pathways, modulate the timing and rates of all three developmental processes. The change in sensitivity in the ovary results from changes in the relative contribution of each pathway to the rates of TF addition and increase in ovary volume before and after critical weight. This work deepens the understanding of how hormones act to modify the sensitivity of organs to environmental conditions, thereby affecting their plasticity (Mendes, 2015).
Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. This study conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-coA production are upregulated, promoting a shift of glucose metabolism towards macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems (Matsuoka, 2017).
Unlike vertically transmitted endosymbionts, which have broad effects on their host's germ line, the extracellular gut microbiota is transmitted horizontally and is not known to influence the germ line. This study provides evidence supporting the influence of these gut bacteria on the germ line of Drosophila melanogaster. Removal of the gut bacteria represses oogenesis, expedites maternal-to-zygotic-transition in the offspring and unmasks hidden phenotypic variation in mutants. It was further shown that the main impact on oogenesis is linked to the lack of gut Acetobacter species, and the Drosophila Aldehyde dehydrogenase (Aldh) gene was identified as an apparent mediator of repressed oogenesis in Acetobacter-depleted flies. The finding of interactions between the gut microbiota and the germ line has implications for reproduction, developmental robustness and adaptation (Elgart, 2016). The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. This study found that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wildtype. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wildtype. Interestingly, even small patches of wildtype follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd) Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. This study identified, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop (not) function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA's histone acetyltransferase module, which does not bind to Expanded or Merlin, is dispensable for migration. Taken together, these results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, that may help guide studies in other systems where USP22 is necessary for cell motility and invasion (Badmos, 2021).
This study reports that Drosophila USP22, encoded by not, is necessary for F-actin polarity and collective cell migration of invasive BCs. Collective BC migration requires actomyosin polymerization and contraction at the cortex around the cluster as it moves over the nurse cell substrate; F-actin is effectively excluded from the center of the cluster where polarity determinants acting via the Hippo complex block the activity of the F-actin regulator Enabled. Not has been reported to regulate the actin cytoskeleton directly by promoting the stability of Scar/WAVE. However, this study did not observe a reduction in Scar levels in not mutant clones, and scar loss of function did not disrupt F-actin polarity. Furthermore, no significant change was observed in the number of actin protrusions following not loss of function. This might be expected if Scar were a target in BCs. Interestingly, scar RNAi weakly suppressed not loss of function, suggesting that accumulation of branched actin, mediated by Scar at BC-BC junctions, may contribute to disrupted cell polarity and impaired migration. The data suggest that not regulates inside-out F-actin polarity by regulating the expression of Hippo signaling components ex and mer, that are direct Not targets, in a yki-independent manner. Reanalysis of ChIP-Seq data from embryos indicates that Not and Ada2b bind other core Hippo pathway components, so expression of multiple components may be affected by loss of SAGA components. However, ex and mer are targets for Not, but not Ada2b, which is largely dispensable for migration. Notably, this study found that overexpression of ex suppressed not1-induced F-actin accumulation at inner BC junctions, consistent with partial restoration of Hippo function and inhibition of Enabled function. It was also observed that cpb overexpression rescued loss of not, again consistent with disruption of Enabled function due to competitive binding of Cpb to F-actin barbed ends and the inhibition of F-actin polymerization at inner BC junctions. Incomplete rescue of not1 with overexpressed ex or cpb means that other parallel downstream targets that contribute to not function may exist. Interestingly, the data suggest that not is dispensable in polar cells for BC migration. It will be interesting to examine whether the requirement for not in Hippo pathway function is limited to situations where the Hippo complex acts in a yki-independent fashion. The nature of putative noncell autonomous signaling mediated by not controlling polar cell number remains to be elucidated, but altered signaling may be an indirect consequence of changes in polarity or via direct changes in the expression of affected signaling molecules (Badmos, 2021).
A striking effect of not loss of function in BCs is the redistribution of Crb from inner to outer BC junctions. When possible effects of this on other polarity determinants were examined, it was found that localization of aPKC to the inside apical junction between BCs was disrupted, consistent with studies showing that Crb, acting together with the Par complex and endocytic recycling machinery, is necessary for ensuring its correct distribution. Mislocalized aPKC generates protrusions at the side and back of BCs, just as were seen in not1 clusters. Why is Crb mislocalized to the cortex of the BC complex? Complementation experiments suggest that this is partially accounted for by loss of expression of the FERM domain proteins Ex and Mer, which in follicle cells act together with Moe to recruit Crb to the apical surface. Moe stabilizes Crb at the apical membrane of epithelia by linking Crb to cortical actin. Although the physical interaction between Moe and Crb may be weak, Moe is an important regulator of dynamic Crb localization because it acts to antagonize interactions between Crb and aPKC at the marginal zone of the apical membrane domain while stabilizing interactions between Crb and the apical surface. Importantly, in BCs, Moe is cortically localized where it organizes a supercellular actin cytoskeleton network and promotes cortical stiffness. An attractive hypothesis, therefore, is that Moe, along with other proteins, is a sink for Crb at the cortex of the BC cluster following loss of Ex and Mer at inner BC junctions in not mutants. When ex was overexpressed, the normal pattern of Crb localization was partially restored in support of there being competitive binding. Interestingly, weak rescue of Crb localization was also observed following Cpb overexpression. This might be because Moe, or other proteins that tether Crb on the outer membrane, is only accessible in the absence of a strong supercellular F-actin cortex and that restoration of cortical F-actin in not1 cpb+ cells displaces Crb. In WT BCs, Crb needs to be constantly moved from the outside membrane in a dynamin- and Rab5-dependent manner. Another possibility therefore, which is not mutually exclusive from the first, is that polarization of the F-actin cytoskeleton is important for correct trafficking of Crb in BCs as it is in follicle cells (Badmos, 2021).
The growth, specification, and migration of cells during tissue development requires precisely regulated patterns of gene expression that depend on numerous cues for temporal and spatial gene activation involving crosstalk with multiple signaling pathways. Strikingly, it has emerged that factors once considered to be ubiquitous regulators of transcription, including the SAGA chromatin-modifying complex, can have specific roles in discrete developmental processes. Although it has been suggested that SAGA is required for all transcribed genes in some contexts, numerous studies have shown that loss of SAGA components affects the expression of only a subset of genes and that different components modulate distinct and overlapping subsets. These differences in expression are likely to explain their different physiological roles; for instance, during female germline development in Drosophila, ada2B affects the expression of many genes and is required for oogenesis, whereas not affects relatively few and is dispensable. Genome-wide ChIP studies indicate that even though both DUB and HAT modules bind the same genes, many of the targets do not require the DUB module for expression, explaining the observed dependencies. These experiments also revealed nonoverlapping sites of chromatin occupancy for the DUB and HAT modules of SAGA in Drosophila, but the significance of differences in transcriptional targeting for cell function had not been established. Notably, in this respect, this study found that the requirement for not in BC migration is not matched by a requirement for HAT components, including ada2b or gcn5. Furthermore, Ada2b has not been found to bind the ex and mer promoters, providing a molecular explanation for not's SAGA-independent role. Importantly, these findings challenge the perceived view that transcriptional roles for not/USP22 are mediated solely by SAGA. This may have broader relevance to situations where USP22, but not other members of SAGA, is associated with human disease states, particularly where cell polarity is frequently disrupted, such as cancer. Current efforts are directed at identifying SAGA-independent factors that facilitate Not's chromatin binding and function (Badmos, 2021).
A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, this study identified that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin's actin bundling activity is required to limit Myosin activation. Surprisingly, this study found that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to reveal that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This understudied means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration (Lamb, 2021).
Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. This study first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. RNAi knockdown of the Drosophila orthologs was performed in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, eight adhesion-related genes were identified that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion (Kotian, 2021).
Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, it was found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, components of Mib2 complexes were identified, includeing E-cadherin and α- and β-catenins, as well as actin regulators. Three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. It is concluded that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms (Trivedi, 2022).
Collective migration plays critical roles in developmental, physiological and pathological processes, and re,quires a dynamic actomyosin network for cell shape change, cell adhesion and cell-cell communication. The dynamic network of mitochondria in individual cells is regulated by mitochondrial fission and fusion, and is required for cellular processes including cell metabolism, apoptosis and cell division. But whether mitochondrial dynamics interplays with and regulates actomyosin dynamics during collective migration is not clear. This study demonstrated that proper regulation of mitochondrial dynamics is critical for collective migration of Drosophila border cells during oogenesis, and misregulation of fission or fusion results in reduction of ATP levels. Specifically, Drp1 is genetically required for border cell migration, and Drp1-mediated mitochondrial fission promotes formation of leading protrusion, likely through its regulation of ATP levels. Reduction of ATP levels by drug treatment also affects protrusion formation as well as actomyosin dynamics. Importantly, this study found that RhoA/ROCK signaling, which is essential for actin and myosin dynamics during border cell migration, could exert its effect on mitochondrial fission through regulating Drp1's recruitment to mitochondria. These findings suggest that RhoA/ROCK signaling may couple or coordinate actomyosin dynamics with mitochondrial dynamics to achieve optimal actomyosin function, leading to protrusive and migratory behavior (Qu, 2022).
Cells migrate collectively through confined environments during development and cancer metastasis. The nucleus, a stiff organelle, impedes single cells from squeezing into narrow channels within artificial environments. However, how nuclei affect collective migration into compact tissues is unknown. This study used border cells in the fly ovary to study nuclear dynamics in collective, confined in vivo migration. Border cells delaminate from the follicular epithelium and squeeze into tiny spaces between cells called nurse cells. The lead cell nucleus transiently deforms within the lead cell protrusion, which then widens. The nuclei of follower cells deform less. Depletion of the Drosophila B-type lamin, Lam, compromises nuclear integrity, hinders expansion of leading protrusions, and impedes border cell movement. In wildtype, cortical myosin II accumulates behind the nucleus and pushes it into the protrusion, whereas in Lam-depleted cells, myosin accumulates but does not move the nucleus. These data suggest that the nucleus stabilizes lead cell protrusions, helping to wedge open spaces between nurse cells (Penfield, 2023).
Chemotaxis drives diverse migrations important for development and involved in diseases, including cancer progression. Using border cells in the Drosophila egg chamber as a model for collective cell migration, this study characterized the role of ArfGAP1 in regulating chemotaxis during this process. ArfGAP1 is required for the maintenance of receptor tyrosine kinases, the guidance receptors, at the plasma membrane. In the absence of ArfGAP1, the level of active receptors is reduced at the plasma membrane and increased in late endosomes. Consequently, clusters with impaired ArfGAP1 activity lose directionality. Furthermore, we found that the number and size of late endosomes and lysosomes are increased in the absence of ArfGAP1. Finally, genetic interactions suggest that ArfGAP1 acts on the kinase and GTPase Lrrk to regulate receptor sorting. Overall, the data indicate that ArfGAP1 is required to maintain guidance receptors at the plasma membrane and promote chemotaxis (Boutet, 2023).
Collective cell migration occurs in various biological processes such as development, wound healing and metastasis. During Drosophila oogenesis, border cells (BC) form a cluster that migrates collectively inside the egg chamber. The Ste20-like kinase Misshapen (Msn) is a key regulator of BC migration coordinating the restriction of protrusion formation and contractile forces within the cluster. This study demonstrates that the kinase Tao acts as an upstream activator of Msn in BCs. Depletion of Tao significantly impedes BC migration and produces a phenotype similar to Msn loss-of-function. Furthermore, it was shown that the localization of Msn relies on its CNH domain, which interacts with the small GTPase Rap2l. These findings indicate that Rap2l promotes the trafficking of Msn to the endolysosomal pathway. When Rap2l is depleted, the levels of Msn increase in the cytoplasm and at cell-cell junctions between BCs. Overall, these data suggest that Rap2l ensures that the levels of Msn are higher at the periphery of the cluster through the targeting of Msn to the degradative pathway. Together, this study identified two distinct regulatory mechanisms that ensure the appropriate distribution and activation of Msn in BCs (Roberto, 2023).
Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Using the Drosophila melanogaster border cells as a model, this study reports that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, it was found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. A molecular mechanism was elucidated that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. It is proposed that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior (Torres, 2023).
Septins (see Drosophila Peanut) self-assemble into polymers that bind and deform membranes in vitro and regulate diverse cell behaviors in vivo. How their in vitro properties relate to their in vivo functions is under active investigation. This study uncovered requirements for septins in detachment and motility of border cell clusters in the Drosophila ovary. Septins and myosin colocalize dynamically at the cluster periphery and share phenotypes but, surprisingly, do not impact each other. Instead, Rho independently regulates myosin activity and septin localization. Active Rho recruits septins to membranes, whereas inactive Rho sequesters septins in the cytoplasm. Mathematical analyses identify how manipulating septin expression levels alters cluster surface texture and shape. This study shows that the level of septin expression differentially regulates surface properties at different scales. This work suggests that downstream of Rho, septins tune surface deformability while myosin controls contractility, the combination of which governs cluster shape and movement (Gabbert, 2023).
The basement membrane (BM) is a specialized extracellular matrix (ECM), which underlies or encases developing tissues. Mechanical properties of encasing BMs have been shown to profoundly influence the shaping of associated tissues. This study used the migration of the border cells (BCs) of the Drosophila egg chamber to unravel a new role of encasing BMs in cell migration. BCs move between a group of cells, the nurse cells (NCs), that are enclosed by a monolayer of follicle cells (FCs), which is, in turn, surrounded by a BM, the follicle BM. Increasing or reducing the stiffness of the follicle BM, by altering laminins or type IV collagen levels, conversely affects BC migration speed and alters migration mode and dynamics. Follicle BM stiffness also controls pairwise NC and FC cortical tension. It is proposed that constraints imposed by the follicle BM influence NC and FC cortical tension, which, in turn, regulate BC migration. Encasing BMs emerge as key players in the regulation of collective cell migration during morphogenesis (Lopez, 2023).
A key regulator of collective cell migration is prostaglandin (PG) signaling. However, it remains largely unclear whether PGs act within the migratory cells or their microenvironment to promote migration. This study used Drosophila border cell migration as a model to uncover the cell-specific roles of two PGs in collective migration. Prior work shows PG signaling is required for on-time migration and cluster cohesion. The PGE (2) synthase cPGES is required in the substrate, while the PGF (2α) synthase Akr1B is required in the border cells for on-time migration. Akr1B acts in both the border cells and their substrate to regulate cluster cohesion. One means by which Akr1B regulates border cell migration is by promoting integrin-based adhesions. Additionally, Akr1B limits myosin activity, and thereby cellular stiffneβ, in the border cells, whereas cPGES limits myosin activity in both the border cells and their substrate. Together these data reveal that two PGs, PGE (2) and PGF (2α), produced in different locations, play key roles in promoting border cell migration. These PGs likely have similar migratory versus microenvironment roles in other collective cell migrations (Mellentine, 2023).
Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, which forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. This work screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. Vinculin was found to work with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. This study has also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. It is concluded that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms (Khaitan, 2023).
Specification of migratory cell fate from a stationary population is complex and indispensable both for metazoan development as well for the progression of the pathological condition like tumor metastasis. Though this cell fate transformation is widely prevalent, the molecular understanding of this phenomenon remains largely elusive. This study employed the model of border cells (BC) in Drosophila oogenesis and identified germline activity of an RNA binding protein, Cup that limits acquisition of migratory cell fate from the neighbouring follicle epithelial cells. As activation of JAK-STAT in the follicle cells is critical for BC specification, these data suggest that Cup, non-cell autonomously restricts the domain of JAK-STAT by activating Notch in the follicle cells. Employing genetics and Delta endocytosis assay, Cup was demonstrated to regulate Delta recycling in the nurse cells through Rab11GTPase thus facilitating Notch activation in the adjacent follicle cells. Since Notch and JAK-STAT are antagonistic, it is proposed that germline Cup functions through Notch and JAK-STAT to modulate BC fate specification from their static epithelial progenitors (Saha, 2023).
Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, this study reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, these studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration (Zhou, 2022).
Drosophila RhoGAP18B was identified as a negative regulator of small GTPase in the behavioral response to ethanol. However, the effect of RhoGAP18B on cell migration is unknown. This study reports that RhoGAP18B regulates the migration of border cells in Drosophila ovary. The RhoGAP18B gene produces four transcripts and encodes three translation isoforms. Different RNAi lines were used to knockdown each RhoGAP18B isoform, and find that knockdown of RhoGAP18B-PA, but not PC or PD isoform, blocks border cell migration. Knockdown of RhoGAP18B-PA disrupts the asymmetric distribution of F-actin in border cell cluster and increases F-actin level. Furthermore, RhoGAP18B-PA may act on Rac to regulate F-actin organization. These data indicate that RhoGAP18B shows isoform-specific regulation of border cell migration (Lei, 2023).
Chemotaxis drives diverse migrations important for development and involved in diseases, including cancer progression. Using border cells in the Drosophila egg chamber as a model for collective cell migration, this study characterized the role of ArfGAP1 in regulating chemotaxis during this process. ArfGAP1 was found to be required for the maintenance of receptor tyrosine kinases, the guidance receptors, at the plasma membrane. In the absence of ArfGAP1, the level of active receptors is reduced at the plasma membrane and increased in late endosomes. Consequently, clusters with impaired ArfGAP1 activity lose directionality. Furthermore, it was found that the number and size of late endosomes and lysosomes are increased in the absence of ArfGAP1. Finally, genetic interactions suggest that ArfGAP1 acts on the kinase and GTPase Lrrk to regulate receptor sorting. Overall, these data indicate that ArfGAP1 is required to maintain guidance receptors at the plasma membrane and promote chemotaxis (Boutet, 2023).
Nuclear hormone receptors have emerged as important regulators of mammalian and Drosophila adult physiology, affecting such seemingly diverse processes as adipogenesis, carbohydrate metabolism, circadian rhythm, stem cell function, and gamete production. Although nuclear hormone receptors Ecdysone Receptor (EcR) and Ultraspiracle (Usp) have multiple known roles in Drosophila development and regulate key processes during oogenesis, the adult function of the majority of nuclear hormone receptors remains largely undescribed. Ecdysone-induced protein 78C (E78), a nuclear hormone receptor closely related to Drosophila E75 and to mammalian Rev-Erb and Peroxisome Proliferator Activated Receptors, was originally identified as an early ecdysone target; however, it has remained unclear whether E78 significantly contributes to adult physiology or reproductive function. To further explore the biological function of E78 in oogenesis, this study used available E78 reporters and created a new E78 loss-of-function allele. E78 was found to be expressed throughout the germline during oogenesis, and was important for proper egg production and for the maternal control of early embryogenesis. E78 was required during development to establish the somatic germline stem cell (GSC) niche; E78 function in the germline promoted the survival of developing follicles. Consistent with its initial discovery as an ecdysone-induced target, there were significant genetic interactions between E78 and components of the ecdysone signaling pathway. Taken together with the previously described roles of EcR, Usp, and E75, these results suggest that nuclear hormone receptors are critical for the broad transcriptional control of a wide variety of cellular processes during oogenesis (Ables, 2015).
Although nuclear hormone receptors are known to play important roles in a wide variety of biological processes, it remains largely unknown whether or how most of the Drosophila nuclear hormone receptors function during oogenesis. This study adds to a growing body of literature demonstrating that nuclear hormone receptors are integral to reproductive function at multiple levels, including reproductive organ development, stem cell function, and gamete development and survival. While it remains unclear how E78 contributes mechanistically to the ecdysone signaling network in the ovary, these studies also highlight the intricate connections between Drosophila nuclear hormone receptors and ecdysone signaling. Given the level of structural and functional conservation between Drosophila and mammalian hormonal signaling pathways, it is proposed that similar connections may exist among diverse mammalian nuclear hormone receptor subtypes. Further studies will be necessary to fully elucidate the molecular networks that tie these pathways together to achieve such important biological regulation (Ables, 2015).
It is interesting to note that each of the ecdysone early response genes studied in the ovary to date (EcR, E74, E75, E78) encode at least two different mRNA isoforms: one long mRNA isoform resulting from splicing of a very long intron separating conserved DNA- and ligand-binding domains, and a shorter isoform that may or may not produce a distinct protein isoform. Previous studies have indicated that Ftz-f1, another ecdysone-regulated nuclear hormone receptor, is also encoded by two different mRNA isoforms: ftz-f1-RA (short isoform) is maternally deposited and required for embryogenesis, while ftz-f1-RB (long isoform) is required at other developmental stages. Future studies investigating whether the various isoforms of ecdysone early-response genes differentially control oogenesis versus embryogenesis will help refine understanding of how a steroid hormone may induce temporal-, developmental-, and cell type-specific effects (Ables, 2015).
While these studies demonstrate a specific requirement for E78 in promoting the survival of germline cysts, the mechanisms by which E78 controls cyst survival remain a topic for further exploration. Indeed, mutants of several ecdysone early-response genes, including EcR, E74, and E75, display similar cyst death near the onset of oocyte vitellogenesis, suggesting that ecdysone signaling promotes a maturation or survival cue during follicle development. Very little is known, however, about the targets of ecdysone signaling during earlier previtellogenic stages. Two recent large-scale screens for regulators of ecdysone-regulated cell death in a haemocyte cell line and in salivary glands may prove useful for identifying targets involved in the decision between cell death and survival. Interestingly, the stage 4/5 cyst death observed in E78δ31 mutants is phenocopied by mutations in insulin and target of rapamycin (TOR) signaling pathway components, including InR, chico, TOR, and S6 kinase Since EcR and E78 appear to functionally cooperate, future studies should test whether EcR and E78 regulate members of the insulin/TOR signaling pathways (or vice versa) to control cyst viability. These basic studies not only will help elucidate the mechanisms by which nuclear hormone receptors control biological processes, but may also add to general understanding of how nuclear hormone receptor signaling is integrated into other endocrine networks to coordinate cell-specific responses with whole-animal physiology (Ables, 2015).
WD40 proteins control many cellular processes via protein interactions. Drosophila Wuho (Wh, a WD40 protein) controls fertility, although the involved mechanisms are unclear. This study shows that Wh promotion of Mei-p26 (a human TRIM32 ortholog) function maintains ovarian germ cell homeostasis. Wh and Mei-p26 are epistatically linked, with wh and mei-p26 mutants showing nearly identical phenotypes, including germline stem cell (GSC) loss, stem-cyst formation due to incomplete cytokinesis between GSCs and daughter cells, and overproliferation of GSC progeny. Mechanistically, Wh interacts with Mei-p26 in different cellular contexts to induce cell type-specific effects. In GSCs, Wh and Mei-p26 promote BMP stemness signaling for proper GSC division and maintenance. In GSC progeny, Wh and Mei-p26 silence nanos translation, downregulate a subset of microRNAs involved in germ cell differentiation and suppress ribosomal biogenesis via dMyc to limit germ cell mitosis. This study also found that the human ortholog of Wh (WDR4) interacts with TRIM32 in human cells. These results show that Wh is a regulator of Mei-p26 in Drosophila germ cells and suggest that the WD40-TRIM interaction may also control tissue homeostasis in other stem cell systems (Rastegari, 2020).
Stem cell self-renewal and differentiation must be balanced for proper tissue homeostasis. This balance is known to be coordinated by transcriptional and post-transcriptional mechanisms, which have not been fully described at a molecular level. An excellent model for studying genetic regulation of the cell fate transition from stem cell to differentiated progeny is the Drosophila ovary, as its germline stem cells (GSCs) and differentiated progeny are well characterized in cell biology. Although the physiology of Drosophila egg production is well described at a cellular level, the molecular regulatory mechanisms are still an area of active investigation (Rastegari, 2020).
Wuho (Wh; meaning 'no progeny' in Chinese) is an evolutionarily conserved protein comprising five WD40 domains (Cheng, 2016; Wu, 2006), which mediate protein-protein interactions. Homologs of Wh have been shown to exert a wide variety of functions via interactions with m7G46 tRNA methyltransferase, Flap endonuclease 1 (FEN1) and Culin-Ring ubiquitin ligase 4. Interestingly, wh mutant males are sterile because their spermatids are not properly elongated to make functional spermatozoa, and female flies are semi-sterile for unknown reasons (Wu, 2006), suggesting that Wh may potentially play an important role in the molecular regulatory circuitry of the GSC lineage (Rastegari, 2020).
Interestingly, the ovarian phenotypes reported in Wh mutants are strikingly similar to those in Mei-p26 mutants. Mei-p26 is a member of the tripartite motif and Ncl-1, HT2A and Lin-41 domain (TRIM- NHL) family of proteins, which is highly conserved among metazoans. TRIM-NHL proteins are known to control developmental transitions through mechanisms such as the promotion of stem cell differentiation by suppressing proliferation. The molecular action of TRIM-NHL proteins is typically ubiquitination and translation silencing via E3 ligase RING domains; meanwhile, the NHL domains mediate protein-protein interactions. In the Drosophila germline, Mei-p26 controls GSC maintenance and differentiation depending on its expression level. However, the regulators of Mei-p26 in the GSC lineage are not known. This study shows that Wh is a key regulator of Mei-p26 and that these proteins function together in multiple contexts to control GSC maintenance and differentiation for germline homeostasis. These results document a potentially generalizable role for WD40 proteins as a bridge between TRIM-NHL proteins and other cellular components, a function that is necessary to balance self-renewal and differentiation in the GSC lineage (Rastegari, 2020).
Homeostatic regulation of stem cells, the very foundation of tissue homeostasis, remains poorly understood at a molecular level. Using the Drosophila GSC lineage as an in vivo model to study stem cell biology, this study found that Wh, a WD40 protein, controls GSC self- renewal and differentiation via Mei-p26, a TRIM-NHL protein. An interaction between the proteins was identified in the ovary, and striking similarities between wh7 and mei-p26 mutants were observed at both phenotypic and molecular levels. Based on these findings and published results regarding Mei-p26 function, it is proposed that, in wild-type GSCs, Wh, Mei-p26 and Nos form a complex to inhibit brat translation, allowing Mad to be stabilized and phosphorylated by BMP signaling. pMad then translocates to the nucleus, where it suppresses transcription of bam, a master regulator of differentiation. On the other hand, Wh also promotes differentiation of GSC progeny by multiple mechanisms. First, Wh, Mei-p26, Bgcn, Sxl and Bam form a complex that binds to the 3' UTR of nos to silence its translation, possibly helping to turn off BMP signaling in the differentiating GSC progeny. Second, Wh interacts with Mei-p26, an E3 ubquitin ligase, to control dMyc protein levels and allow proper ribosomal biogenesis. Third, the interaction between Wh and Mei-p26 also limits expression of a subset of microRNAs, which may contribute to differentiation. These functions of Wh appear to be especially important for the last step of cytokinesis (abscission), which is completed between the GSC and its daughter cell after early G2 phase, as revealed by closure of the ring canal (Rastegari, 2020).
In wh mutant GSCs, brat translation is not suppressed, decreasing the level of pMad and increasing the expression of the differentiation factor Bam. This sequence of events causes premature differentiation of GSCs and leads to GSC loss, consistent with the known roles of Brat and Bam in germ cell differentiation. Mutation of brat or bam increases GSC number, whereas overexpression of brat or bam in germ cells causes germ cell depletion by forcing GSC differentiation. Mutation of wh in germ cells also results in incomplete abscission between GSCs and daughter cells, leaving open ring canals that create stem-cysts. Although this study did not determine the molecular mechanism by which Wh controls GSC abscission, removing a copy of bam significantly reduced stem-cyst number in wh mutant germaria, which suggests a role for Bam in Wh control of GSC abscission. In wh mutant germaria, stem-cysts simultaneously express Nos (a GSC maintenance factor) and Bam; thus, the growths display characteristics of both GSCs and daughter cells. In addition, ribosomal biogenesis is promoted via upregulated dMyc and drives germ cell overproliferation. Lastly, some differentiation-associated microRNAs are increased in the mutant ovaries, although their functions are not yet clear. It is not known whether a defect in meiosis is a consequence of overproliferative wh mutant germ cells, or whether Wh has a separate role in meiosis (Rastegari, 2020).
The results show that Wh is required for Mei-p26 function in germ cell homeostasis. However, it is unclear whether Wh directly interacts with Mei-p26 and which domains in the two proteins mediate the interaction. It is possible that Wh serves as a bridge for Mei-p26 to interact with its known partners. This study observed an interaction between human orthologs of Wh (WDR4) and Mei-p26 (TRIM32), suggesting that the interaction is evolutionarily conserved. Thus, the interaction between WD40 and TRIM-NHL proteins may be crucial for stem cell regulation in other organisms (Rastegari, 2020).
The last step in cell division, cytokinesis, is completed by abscission, which physically separates the two daughter cells. Cytokinesis starts by ingression of the cleavage furrow, constricting the plasma membrane onto the spindle midzone to form an electron- dense structure, the midbody, which comprises a thin membrane channel bridging two nascent daughter cells. The stem-cyst forms owing to a failure of GSCs to separate from daughter cells. Two possible mechanisms may produce such an abscission failure. First, a stem-cell-specific defect may prevent GSC-CB abscission. Second, GSCs may exhibit characteristics of differentiating cells that cause them to adopt incomplete cell cytokinesis programs. In addition to controlling chromosome orientation and segmentation, Aurora B is known to intrinsically regulate the timing of cell abscission, including in Drosophila female GSCs. During abscission, Aurora B in GSCs is targeted to the midbody and triggers membrane abscission via Endosomal sorting complex required for transport III (ESCRT-III) machinery. Aurora B negatively controls ESCRT-III, i.e. when Aurora B is active, ESCRT-III activity is low and abscission is delayed, and vice versa. It has also been shown that ribosomal biogenesis coordinates with ESCRT-III in GSCs to promote GSC abscission. Increasing Aurora B activity or disrupting ESCRT-III generates stem-cysts with germ cells that undergo synchronous division, yielding 32 germ cells in most egg chambers. However, wh mutant germ cells within stem-cysts divide asynchronously with elevated ribosomal biogenesis, and wh mutant egg chambers carry various numbers of germ cells. In addition, in wh mutant stem-cysts,
decreased pMad expression, upregulated Bam expression and branched fusomes are all hallmarks of differentiating cysts. These results suggest that wh mutant stem-cysts may adopt the abscission program of differentiating cysts. Interestingly, removing a copy of bam from wh mutants or from shrub (a subunit of ESCRT-III) mutants partially rescues stem-cysts, suggesting that GSC abscission is coupled with cell fate. Further studies will be required to understand the molecular targets of Wh that control GSC abscission, and how GSCs and differentiating cysts acquire different abscission programs. Nevertheless, this study has shown that Wh participates with Mei-p26 to regulate fate determination in stem cells and daughter cells. This novel interaction may be conserved in other species and introduces the idea that WD40 proteins may participate with TRIM-NHL proteins in cell fate decision (Rastegari, 2020).
The insect steroid hormone ecdysone is a key regulator of oogenesis in Drosophila melanogaster and many other species. Despite the diversity of cellular functions of ecdysone in oogenesis, the molecular regulation of most ecdysone-responsive genes in ovarian cells remains largely unexplored. A functional screen was performed using the UAS/Gal4 system to identify non-coding cis-regulatory elements within well-characterized ecdysone-response genes capable of driving transcription of an indelible reporter in ovarian cells. Using two publicly available transgenic collections (the FlyLight and Vienna Tiles resources), 62 Gal4 drivers were tested corresponding to ecdysone-response genes EcR, usp, E75, br, ftz-f1 and Hr3. 31 lines were observed that were sufficient to drive a UAS-lacZ reporter in discrete cell populations in the ovary. Reporter expression was reproducibly observed in both somatic and germ cells at distinct stages of oogenesis, including those previously characterized as critical points of ecdysone regulation. These studies identified several useful new reagents, adding to the UAS/Gal4 toolkit available for genetic analysis of oogenesis in Drosophila. Further, this study provides novel insight into the molecular regulation of ecdysone signaling in oogenesis (McDonald, 2019).
The effect of diet on reproduction is well documented in a large number of organisms; however, much remains to be learned about the molecular mechanisms underlying this connection. The Drosophila ovary has a well described, fast and largely reversible response to diet. Ovarian stem cells and their progeny proliferate and grow faster on a yeast-rich diet than on a yeast-free (poor) diet, and death of early germline cysts, degeneration of early vitellogenic follicles and partial block in ovulation further contribute to the approximately 60-fold decrease in egg laying observed on a poor diet. Multiple diet-dependent factors, including insulin-like peptides, the steroid ecdysone, the nutrient sensor Target of Rapamycin, AMP-dependent kinase, and adipocyte factors mediate this complex response. This describe the results of a visual screen using a collection of green fluorescent protein (GFP) protein trap lines to identify additional factors potentially involved in this response. In each GFP protein trap line, an artificial GFP exon is fused in frame to an endogenous protein, such that the GFP fusion pattern parallels the levels and subcellular localization of the corresponding native protein. Fifty-three GFP-tagged proteins were identified that exhibit changes in levels and/or subcellular localization in the ovary at 12-16 hours after switching females from rich to poor diets, suggesting them as potential candidates for future functional studies (Hsu, 2017).
Ovariole number has a direct role in the number of eggs produced by an insect, suggesting that it is a key morphological fitness trait. Many studies have documented the variability of ovariole number and its relationship to other fitness and life-history traits in natural populations of Drosophila. However, the genes contributing to this variability are largely unknown. A genome-wide association study of ovariole number was conducted in a natural population of flies. Using mutations and RNAi-mediated knockdown, the effects of twenty-four candidate genes on ovariole number was confirmed, including a novel gene, anneboleyn (formerly CG32000), that impacts both ovariole morphology and numbers of offspring produced. Pleiotropic genes were identified that regulated ovariole number traits and sleep and activity behavior. While few polymorphisms overlapped between sleep parameters and ovariole number, thirty-nine candidate genes were nevertheless in common. The effects of seven genes on both ovariole number and sleep were verified: bin3, blot, CG42389, kirre, slim, VAChT, and zfh1. Linkage disequilibrium among the polymorphisms in these common genes was low, suggesting that these polymorphisms may evolve independently (Lobell, 2017).
Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. This study shows that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, this study confirmed in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. This study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation (Bose, 2022).
The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. This study reports that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte (Tanaka, 2021).
Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, this study showed that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, these results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, these findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context (Eichler, 2023).
The finding that founder granules appear to be agglomerates of osk RNPs provides new insight into the process by which osk accumulates at the posterior of the oocyte during late stages of oogenesis. osk is transported in RNPs containing 2–4 transcripts. Recent work has shown that these RNPs are initially liquid-like condensates but they rapidly mature to a non-dynamic, solid state that prevents incorporation of additional mRNA molecules. Inducing a more liquid-like state results in formation of large, dynamic condensates at the posterior of late-stage oocytes that subsequently detach, indicating that the solid state is necessary for proper founder granule assembly and anchoring. Th4 observation that founder granules contain multiple physically distinct osk RNPs packed together is consistent with the solid-like properties of these RNPs and indicates that they do not form through the collapse of transport RNPs into larger condensates but rather through an aggregative process. This mechanism contrasts with Drosophila germ granules, whereby pre-formed protein condensates are populated by RNPs containing single transcripts, which then self-assemble within the granules to form homotypic clusters. What limits the agglomeration of osk RNPs into founder granules to the posterior of the oocyte remains unclear. RNP-RNP associations may be fostered by the high posterior concentration of osk RNPs achieved previously by kinesin-dependent transport. Since proteins can partition into osk RNPs after their transition to a solid-like state, proteins recruited to the germ plasm, perhaps by Osk protein itself, could mediate this behavior. Intriguingly, zebrafish germ plasm mRNAs form homotypic RNPs that aggregate into compact structures while retaining their distinct spherical appearance. This similarity with founder granules suggests that agglomeration may be a more generalized mechanism for mRNA compartmentalization (Eichler, 2023).
The function of the earlier acting EJC/SOLE complex in late-phase osk localization was interrogated, and the SOLE, but not the adjacent EJC, was found to be required. Whereas the function of the SOLE in the earlier localization phase relies only on the structure of the proximal stem, both the sequence of the proximal stem and its structure are important for late-phase localization. How the SOLE collaborates with the EJC to promote kinesin-dependent osk motility and what, if any regulatory factor interacts with it are not yet known. The sequence-dependence of the SOLE and lack of requirement for the EJC in late-phase localization suggests a different mode of action, possibly through the binding of a different protein to the proximal stem and recruitment of new RNP components or through RNA-RNA interactions. The change in ovarian physiology with the onset of nurse cell dumping could lead to an exchange of proteins associated with osk, to inhibit kinesin-dependent motility and promote posterior agglomeration (Eichler, 2023).
The failure of osk-K10_3'UTR mRNA to localize at late stages of oogenesis despite the presence of the SOLE indicates that similarly to the earlier phase, late-phase localization depends on both the SOLE and the 3' UTR. This dependence on the 3' UTR for the late accumulation of osk is not for the purpose of hitchhiking, and by swapping the osk and nos 3' UTRs it was shown that the osk 3' UTR specifies association of osk RNPs in founder granules independently of the SOLE. This function of the osk 3' UTR may be conferred by the same 3' UTR-binding proteins that control the formation and/or initial localization of osk transport RNPs and remain associated with osk at the posterior pole, such as Bru1, Stau, or Hrp48. For example, a prion-like domain in Bru1 required for formation of osk transport RNPs could also mediate self-association of these RNPs when they come in contact at the posterior pole. Likewise, mammalian Stau has the propensity to form cytoplasmic aggregates. The requirements for Bru1 and Stau in the earlier phase of osk localization make it difficult to test this idea, however. Additionally, the osk 3' UTR may function to prevent co-condensation of osk RNPs with germ granules through the recruitment of proteins like Hrp48, which maintains the solid-like properties of osk RNPs (Eichler, 2023).
Results from swapping the osk and nos 3' UTRs also suggest that either osk 5' UTR and/or coding sequences other than the SOLE contribute to maintaining osk RNPs in founder granules. Since multivalent interactions are typically required for inclusion of components in phase separated condensates, it is not surprising that binding of founder granule components to multiple sites within osk would be required for the integrity of these granules. Further dissection of the sequence requirements and identification of interacting factors will be necessary to define the mechanisms by which the various osk elements accomplish the different tasks (Eichler, 2023).
The process by which osk mRNA achieves its posterior localization is remarkably complex and labor intensive, involving distinct machineries for transport into the oocyte, movement to the posterior pole during stages 8 to 10, and further accumulation during late stages of oogenesis. Given the dependence of embryonic abdominal patterning and germ cell formation on the amount of osk mRNA localized during oogenesis, the reliance on numerous distinct contributions to osk localization likely provides robustness to processes governing the targeting of osk RNPs to the right location and the accumulation of sufficient osk there. Moreover, the distinct process of assembling founder granules ensures that osk mRNA remains separated from germ granules to promote its degradation in the embryonic germ plasm and minimize its inheritance by pole cells (Eichler, 2023).
The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center, integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. This study use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, this study found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the microtubule organizing center, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event (Kenny, 2021).
Study of the timing and location for mRNA translation across model systems has begun to shed light on molecular events fundamental to such processes as intercellular communication, morphogenesis, and body pattern formation. In D. melanogaster, the posterior mRNA determinant, oskar, is transcribed maternally but translated only when properly localized at the oocyte's posterior cortex. Two effector proteins, Bruno1 and Cup, mediate steps of oskar mRNA regulation. The current model in the field identifies Bruno1 as necessary for Cup's recruitment to oskar mRNA and indispensable for oskar's translational repression. This study now reports that this Bruno1-Cup interaction leads to precise oskar mRNA regulation during early oogenesis and, importantly, the two proteins mutually influence each other's mRNA expression and protein distribution in the egg chamber. These factors were shown to be stably associated with oskar mRNA in vivo. Cup associates with oskar mRNA without Bruno1, while surprisingly Bruno1's stable association with oskar mRNA depends on Cup. It was demonstrated that the essential factor for oskar mRNA repression in early oogenesis is Cup, not Bruno1. Furthermore, it was found that Cup is a key P-body component that maintains functional P-body morphology during oogenesis and is necessary for oskar mRNA's association with P-bodies. Therefore, Cup drives the translational repression and stability of oskar mRNA. These experimental results point to a regulatory feedback loop between Bruno 1 and Cup in early oogenesis that appears crucial for oskar mRNA to reach the posterior pole and its expression in the egg chamber for accurate embryo development (Bayer, 2023).
Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. This study reports that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, this study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila. Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. The study shows that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis (Wong, 2017).
Recent studies have uncovered a class of stable intronic sequence RNAs (sisRNAs) that are derived from the introns post splicing. sisRNAs are present in various organisms such as viruses, yeast, Drosophila, Xenopus, and mammals. Studies in Drosophila and mammalian cells suggest that sisRNAs function in regulating the expression of their parental genes (host genes where they are derived from) via positive or negative feedback loops. In yeast, sisRNAs are involved in promoting robustness in response to stress, while in Drosophila, sisRNAs have been shown to be important for embryonic development. However, very little is understood about the biological functions of sisRNAs in terms of regulating cellular processes such as differentiation, proliferation, and cell death (Wong, 2017).
The Drosophila genome encodes for several double-stranded RNA (dsRNA) binding proteins that localize to the nucleus. Most of them have been found to regulate specific RNA-mediated processes such as RNA editing, X chromosome activation, and miRNA biogenesis. The Disco-interacting protein 1 (DIP1) is a relatively less characterized dsRNA binding protein that has been implicated in anti-viral defense and localizes to the nucleus as speckles. Otherwise, not much is known about the biological processes regulated by DIP1 (Wong, 2017 and references therein).
This paper show that the regulation of a Drosophila sisRNA sisR-1 by DIP1 is important for keeping female germline stem cell homeostasis in place. DIP1 is shown to regulate INE-1 sisRNAs and localizes to a previously undescribed nuclear body around the fourth chromosomes, called the satellite body. The regulation of sisR-1, which is not on the fourth chromosome, by DIP1 presumably does not occur in the satellite bodies (Wong, 2017).
The results reveal the importance of the regulation of sisRNA activity/expression in GSC-niche occupancy. It is proposed that the sisR-1 axis maintains GSCs in the niche, however, uncontrolled accumulation of sisR-1 due to its unusual stability can lead to increase number of GSCs at the niche. DIP1 in turn limits the build-up of sisR-1 to maintain ~2 GSCs per niche. GSC-niche occupancy is highly regulated by homeostatic mechanisms via negative feedback loops at the cellular and molecular levels. Misregulation of the niche may pose a problem as it allows for a greater chance of GSCs to accumulate mutations that may lead to tumor formation. On the other hand, mechanisms that promote GSC-niche occupancy may be important to facilitate the replenishment of GSCs during aging. Understanding the control of stem cell-niche occupancy will provide important insights to reproduction, cancer, and regenerative medicine (Wong, 2017).
In a large-scale RNAi screen for genes that regulate GSC self-renewal and differentiation, rga was identified as a gene required for GSC differentiation. How rga regulates GSC self-renewal is currently unknown but the current data suggest that GSC-niche adhesion and Mei-P26 are involved. The rga gene encodes for the NOT2 protein in the CCR4 deadenylase complex. Surprisingly, studies have shown that other components of the CCR4 complex such as CCR4, Not1, and Not3 function in promoting GSC maintenance. Interestingly, Twin has been proposed to function with distinct partners to mediate different effects on GSC fates. This suggests that other components such as CCR4 can also have additional functions outside the CCR4-NOT deadenylase complex in mediating GSC maintenance, thus affecting GSCs in opposite ways to Rga (Wong, 2017).
This study puts forward a proposed model for sisR-1-mediated silencing. It is hypothesized that folded sisR-1 harboring a 3' tail may form a ribonucleoprotein complex, which confers its stability, and allows scanning for its target via its 3' tail. Binding of the 3' tail to the target may promote local unwinding of sisR-1 as the 3' end of ASTR invades to form a more stable 76 nt duplex. This study shows that, in principle, it is possible to design a chimeric sisRNA to target a long ncRNA of interest such as rox1. In future, sisRNA can be potentially developed as tools to regulate nuclear RNAs of interest. Clearly, the efficiency and specificity of sisRNA-mediated silencing need to be optimized. Because sisRNA-mediated target degradation requires a more extensive base-pairing between sisRNA and the target, the chances of off-target effects ought to be lower than siRNAs and antisense oligonucleotides. In broader terms, this study provides a paradigm, which encourages exploration of whether other sisRNAs or ncRNAs utilize a similar silencing strategy as sisR-1 (Wong, 2017).
This study describes a nuclear body (named satellite body) that associates with the fourth chromosomes. Satellite body adds to an existing group of nuclear bodies (nucleolus, HLB, and pearl) that associate with specific genomic loci. It is generally believed that formation of such nuclear bodies correlates with a high concentration of RNA transcribed from the tandemly repeated gene loci. The formation of satellite bodies around the fourth chromosomes probably reflects a high concentration of DIP1 in regulating INE-1 sisRNAs transcribed there. The formation of satellite bodies may be promoted by the high concentration of INE-1 sisRNAs transcribed on the fourth chromosomes, and may facilitate the decay of INE-1 sisRNAs . It is speculated that in the nucleoplasm, DIP1 that does not form observable satellite bodies is sufficient to regulate sisRNAs such as sisR-1 transcribed from other chromosomes. Since DIP1 is a dsRNA binding protein, it may bind to mature sisRNAs to destabilize them. It may do so by recruiting RNA degradation factors (such as nuclear exosomes) or introducing RNA modification to 'mark' sisRNAs for degradation. In future, it will be important to identify more components of the satellite bodies and their dynamics during differentiation and in response to stimuli in order to better understand the molecular mechanism of sisRNA metabolism (Wong, 2017).
It is well known that cyclinB3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, Drosophila cycB3 was shown to plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans (Chen, 2018).
The cycB3 gene is evolutionarily-conserved among higher eukaryotic organisms examined, from insects to mammalians. The cycB3 protein is present as Cyclin A and B (two other B-type Cyclins) in mitotically-proliferating cells, and is involved in the regulation of mitosis, where it cooperates with Cyclin A and B. It is reported that Cyclin A and B are involved in the regulation of ovarian GSC maintenance in Drosophila. Earlier observation showed that cycB32 homozygous mutant females partially exhibit thinned ovaries. Given reports on Cyclin activity in stem cells, the thinned ovaries prompted a further exploration of the potential involvement of cycB3 in the maintenance of germline stem cells, in the Drosophila ovary. The phenotypic assays indicate that a cycB3 deficiency leads to GSC loss with ageing. The rescue assays and genetic mosaic analyses convincingly suggest that CycB3 functions as an intrinsic factor for controlling the fate of GSC (Chen, 2018).
Previous studies have discovered that the Dpp/Bam pathway is the essential signaling pathway for maintaining GSCs in the Drosophila ovary. The bam gene is a key switch in regulating the fate of GSC. Combining these results, a model is proposed to explain how CycB3 is involved in regulation of GSC/CB fate determination. In GSCs, the data show that CycB3 is not involved in Dpp-mediated bam transcriptional silencing. The cycB3 deficiency triggers GSC pre-differentiation and eventually causes its loss phenotype. In CBs, the bam gene exhibits a high expression level, due to loss of the inhibition by Dpp signaling, and the Bam protein can promote CB differentiation. Genetic interaction analyses strongly shows that cycB3 function is positioned upstream of Bam action in CBs. The excess cycB3 come from cycB3 overexpression, which specifically suppresses CB differentiation, probably through repressing the activity of Bam. However, what are the factors that functionally position upstream of cycB3 in CBs of Drosophila ovary? This still remains elusive (Chen, 2018).
It is reported that cycB3 promotes metaphase–anaphase transition in Drosophila embryos. The current data show that overexpression of cycB3 fails to increase the number of GSCs, suggesting that the excess CycB3 may fail to influence transition into the GSC system, whereas the excess CycB3 is sufficient to delay CB differentiation. The underlying molecular mechanism might be due to the fact that the increased CycB3 activity is sufficient to enhance CB proliferation, by promoting metaphase-anaphase transition (Chen, 2018).
Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. This study used quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells (Dai, 2017).
Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential to orientate the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a GFP fusion of the spectrosome component Par-1, this study followed the complete spectrosome cycle throughout GSC division and quantified the relative duration of the different spectrosome shapes. It was also determined that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis, and the continuous addition of new material to the GSC and cystoblast spectrosomes was observed. Next, the Fly-FUCCI tool was used to define in live and fixed tissues that GSCs have a shorter G1 compared to the G2 phase. The observation of centrosomes in dividing GSCs allowed determination that centrosomes separate very early in G1, prior to centriole duplication. Furthermore, this study showed that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, this study demonstrated that the asymmetric division of GSCs is not an intrinsic property of these cells, since the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche (Villa-Fombuena, 2021).
The Drosophila female germline stem cell (GSC) niche
provides an excellent model for understanding the stem cell niche in
vivo. The GSC niche is composed of stromal cells that provide growth
factors for the maintenance of GSCs and the associated extracellular
matrix (ECM). Although the function of stromal cells/growth factors has
been well studied, the function of the ECM in the GSC niche is largely
unknown. This study investigated the function of syndecan and perlecan,
molecules of the heparan sulfate proteoglycan (HSPG) family, as the
main constituents of the ECM. Both of these genes were expressed in
niche stromal cells, and knockdown of them in stromal cells decreased
GSC number, indicating that these genes are important niche components.
Interestingly, genetic analysis revealed that the effects of syndecan and perlecan on the maintenance of GSC were distinct. While the knockdown of perlecan in the GSC niche increased the number of cystoblasts, a phenotype
suggestive of delayed differentiation of GSCs, the same was not true in
the context of syndecan. Notably, the overexpression of syndecan and perlecan did not cause an expansion of the GSC niche, opposing the results reported in the context of glypican,
another HSPG gene. Altogether, these data suggest that HSPG genes
contribute to the maintenance of GSCs through multiple mechanisms, such
as the control of signal transduction, and ligand
distribution/stabilization. Therefore, this study paves the way for a
deeper understanding of the ECM functions in the stem cell niche (Hayashi, 2021).
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 FruM). 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).
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 utilize a genetically tractable model for stem cell survival in the Drosophila gonad to screen drug candidates and probe chemical-genetic interactions. This study employed three levels of small molecule screening: (1) a medium-throughput primary screen in male germline stem cells (GSCs), (2) a secondary screen with irradiation and protein-constrained food in female GSCs, and (3) 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. Further, these small molecules were tested for chemical-genetic interactions in the germline, and the NF-κB pathway was identified 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).
Limiting BMP signalling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signalling in both the Drosophila embryo and wing disc is the Brinker (Brk) transcription factor, yet the expression and potential role of brk in the germarium has not previously been described. This study found that brk expression requires a promoter-proximal element (PPE), to both support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to precisely regulate brk levels. Using PPE mutants as well as tissue specific RNAi and overexpression, this study shows that altering brk expression within either the soma or germline affects germ cell homeostasis. Remarkably, it was found that Decapentaplegic (Dpp), the main BMP ligand and Brk's canonical antagonist, is upregulated by Brk in the escort cells of the germarium demonstrating that Brk can positively regulate this pathway (Dunipace, 2022).
The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). This study investigated contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies (Kitzman, 2021).
The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. This study reports that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, these observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis (Russell, 2021).
In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. Mating-induced increase in female GSCs of D. melanogaster were extensively studied at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model (Hoshino, 2021).
Clonal dominance arises when the descendants (clones) of one or a few founder cells contribute disproportionally to the final structure during collective growth. In contexts such as bacterial growth, tumorigenesis, and stem cell reprogramming, this phenomenon is often attributed to pre-existing propensities for dominance, while in stem cell homeostasis, neutral drift dynamics are invoked. The mechanistic origin of clonal dominance during development, where it is increasingly documented, is less understood. This study investigated this phenomenon in the Drosophila melanogaster follicle epithelium, a system in which the joint growth dynamics of cell lineage trees can be reconstructed. This study demonstrated that clonal dominance can emerge spontaneously, in the absence of pre-existing biases, as a collective property of evolving excitable networks through coupling of divisions among connected cells. Similar mechanisms have been identified in forest fires and evolving opinion networks; the spatial coupling of excitable units explains a critical feature of the development of the organism, with implications for tissue organization and dynamics (Alsous, 2021).
Despite their medical and economic relevance, it remains largely unknown how suboptimal temperatures affect adult insect reproduction. This study reports an in-depth analysis of how chronic adult exposure to suboptimal temperatures affects oogenesis using the model insect Drosophila melanogaster. In adult females maintained at 18°C (cold) or 29°C (warm), relative to females at the 25°C control temperature, egg production was reduced through distinct cellular mechanisms. Chronic 18°C exposure improved germline stem cell maintenance, survival of early germline cysts and oocyte quality, but reduced follicle growth with no obvious effect on vitellogenesis. By contrast, in females at 29°C, germline stem cell numbers and follicle growth were similar to those at 25°C, while early germline cyst death and degeneration of vitellogenic follicles were markedly increased and oocyte quality plummeted over time. Finally, this study also showed that these effects are largely independent of diet, male factors or canonical temperature sensors. These findings are relevant not only to cold-blooded organisms, which have limited thermoregulation, but also potentially to warm-blooded organisms, which are susceptible to hypothermia, heatstroke and fever (Gandara, 2022).
In Drosophila ovary, niche is composed of somatic cells, including terminal filament cells (TFCs), cap cells (CCs) and escort cells (ECs), which provide extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Niche establishment begins in larval stages when terminal filaments (TFs) are formed, but the underlying mechanism for the development of TFs remains largely unknown. This study reports that transcription factor longitudinals lacking (Lola) is essential for ovary morphogenesis. Lola protein was expressed abundantly in TFCs and CCs, although also in other cells, and lola was required for the establishment of niche during larval stage. Importantly, it was found that knockdown expression of lola induced apoptosis in adult ovary, and that lola affected adult ovary morphogenesis by suppressing expression of Regulator of cullins 1b (Roc1b), an apoptosis-related gene that regulates caspase activation during spermatogenesis. These findings significantly expand understanding of the mechanisms controlling niche establishment and adult oogenesis in Drosophila (Zhao, 2022).
Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, mutants were generated for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed analysis of the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. These observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture (Knudsen, 2023).
Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. The data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. It is speculated that this pathway is conserved across organisms to regulate oocyte development and promote fertility (Giedt, 2023).
Having intact epithelial tissues is critical for embryonic development and adult homeostasis. How epithelia respond to damaging insults or tissue growth while still maintaining intercellular connections and barrier integrity during development is poorly understood. The conserved small GTPase Rap1 is critical for establishing cell polarity and regulating cadherin-catenin cell junctions. This study identified a new role for Rap1 in maintaining epithelial integrity and tissue shape during Drosophila oogenesis. loss of Rap1 activity disrupted the follicle cell epithelium and the shape of egg chambers during a period of major growth. Rap1 was required for proper E-Cadherin localization in the anterior epithelium and for epithelial cell survival. Both Myo-II and the adherens junction-cytoskeletal linker protein α-Catenin were required for normal egg chamber shape but did not strongly affect cell viability. Blocking the apoptotic cascade failed to rescue the cell shape defects caused by Rap1 inhibition. One consequence of increased cell death caused by Rap1 inhibition was the loss of polar cells and other follicle cells, which later in development led to fewer cells forming a migrating border cell cluster. These results thus indicate dual roles for Rap1 in maintaining epithelia and cell survival in a growing tissue during development (Messer, 2023).
During Drosophila oogenesis, somatic follicle cells (FCs) differentiate to secrete components of the eggshell. Before secretion, the epithelium reorganizes to shape eggshell specializations, including border FC collective cell migration and later dorsal formation. These FC movements provide valuable insights into collective cell migration. However, little is known about centripetal migration, which encloses the oocyte after secretion has begun. Centripetal migration begins with apical extension of a few FCs that move away from the basement membrane to invade between germ cells. This study defines a timeline of reproducible milestones, using time-lapse imaging of egg chamber explants. Inward migration occurs in two phases. First, leading centripetal FCs ingress, extending apically over the anterior oocyte, and constricting basally. Second, following FCs move collectively toward the anterior, then around the corner to move inward with minimal change in aspect ratio. E-cadherin was required in leading centripetal FCs for their normal ingression, assessed with homozygous shotgun mutant or RNAi knockdown clones; ingression was influenced non-autonomously by mutant following FCs. This work establishes centripetal migration as an accessible model for biphasic E-cadherin-adhesion-mediated collective migration (Parsons, 2023).
RNA binding proteins (RBPs) play a fundamental role in the post-transcriptional regulation of gene expression within the germline and nervous system. This is underscored by the prevalence of mutations within RBP-encoding genes being implicated in infertility and neurological disease. Previous work described roles for the highly conserved RBP Caper in neurite morphogenesis in the Drosophila larval peripheral system and in locomotor behavior. However, caper function has not been investigated outside the nervous system, although it is widely expressed in many different tissue types during embryogenesis. This study describes novel roles for Caper in fertility and mating behavior.Caper is expressed in ovarian follicles throughout oogenesis but is dispensable for proper patterning of the egg chamber. Additionally, reduced caper function, through either a genetic lesion or RNA interference-mediated knockdown of caper in the female germline, results in females laying significantly fewer eggs than their control counterparts. Moreover, this phenotype is exacerbated with age. caper dysfunction also results in partial embryonic and larval lethality. Given that caper is highly conserved across metazoa, these findings may also be relevant to vertebrates (Tixtha, 2022).
">CTP synthase (CTPS) forms a filamentous structure termed the cytoophidium in all three domains of life. The female reproductive system of Drosophila is an excellent model for studying the physiological function of cytoophidia. This study used CTPS(H355A), a point mutation that destroys the cytoophidium-forming ability of CTPS, to explore the in vivo function of cytoophidia. In CTPS(H355A) egg chambers, the ingression and increased heterogeneity of follicle cells was observed. In addition, it was found that the cytoophidium-forming ability of CTPS, rather than the protein level, is the cause of the defects observed in CTPS(H355A) mutants. To sum up, these data indicate that cytoophidia play an important role in maintaining the integrity of follicle epithelium (Wang, 2023).
Apicobasal cell-polarity loss is a founding event in Epithelial-Mesenchymal Transition (EMT) and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, single-cell RNA sequencing was performed of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, it was discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, these findings describe the comprehensive transcriptome of cells within the follicle-cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis (Chatterjee, 2022).
Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, understanding of non-apoptotic caspase functions remains limited. This study showed that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, the key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions (Galasso, 2023).ccxv
Shaping of developing organs requires dynamic regulation of force and resistance to achieve precise outcomes, but how organs monitor tissue mechanical properties is poorly understood. This study shows that in developing Drosophila follicles (egg chambers), a single pair of cells performs such monitoring to drive organ shaping. These anterior polar cells secrete a matrix metalloproteinase (MMP) that specifies the appropriate degree of tissue elongation, rather than hyper- or hypo-elongated organs. MMP production is negatively regulated by basement membrane (BM) mechanical properties, which are sensed through focal adhesion signaling and autonomous contractile activity; MMP then reciprocally regulates BM remodeling, particularly at the anterior region. Changing BM properties at remote locations alone is sufficient to induce a remodeling response in polar cells. It is proposed that this small group of cells senses both local and distant stiffness cues to produce factors that pattern the organ's BM mechanics, ensuring proper tissue shape and reproductive success (Ku, 2023).
It is now appreciated that anisotropic ECM organization can drive tissue shape in both vertebrates and invertebrates, playing critical roles from early embryos to developing organs to adult reproduction. Yet, how specifically patterned BMs are generated and maintained is poorly understood. This work provides a model for how specialized cells can specify local and long-range mechanical properties including by regulating BM texture. The data suggest that as elongation of the Drosophila follicle initiates, polar cells (PCs) at the anterior pole use focal adhesion signaling to perceive an initial low degree of graded BM stiffness, and then they tune MMP1 activity to promote the formation of BM fibrils that can confer anisotropic tensile strength. This process must be carefully regulated to ensure that it does not result in hyperelongation. Interestingly, the BM also provides reciprocal biomechanical feedback to regulate the PCs. As the follicle stiffens, PCs experience more focal adhesion signaling that then moderates MMP production. These mechanical monitoring and modifying processes encompass not just the local anterior environment but extend more broadly through the tissue. Indeed, a striking finding of this work is that BM changes in the follicle center are sensed at a distance by focal adhesion signaling restricted to the PC, although how these changes are transmitted ia unknoqn. Overall, this work uncovers a mechanism by which a growing organ morphogenetically self-organizes, dynamically regulating its own BM patterning to ensure appropriate shape (Ku, 2023).
The effector molecule through which PCs specify non-autonomous BM organization is the fly matrix metalloprotease MMP1. When PC MMP1 levels increase, ColIV fibril density increases, the BM becomes more resistant to mechanical failure during expansion, and tissue elongation along the AP axis increases; the opposite occurs when PC MMP1 levels are experimentally reduced. This unexpected result is contradictory to the canonical ECM-degrading role of MMPs. However, it is consistent with recent studies and earlier work establishing that BM ColIV in developing organs is highly dynamic, and its remodeling can be promoted by the activity of MMP1 and other metalloproteinases (Ku, 2023).
In all of these cases, the direct mechanism of ColIV regulation has not been discerned. The data show that MMP1 does not control overall ColIV levels; moreover, MMP1 is unlikely to promote formation of de novo BM fibrils that are organized prior to secretion (Ku, 2023).
Instead, they suggest that MMP1 activity results in altered extracellular organization of ColIV, specifically promoting remodeling from the conventional isotropic basal lamina into a fibril network whose characteristics include increased density. The impact of MMP1 on mechanical properties is most strong at the anterior but ramifies further into the organ. The difference between MMP1’s limited anterior range and its long-range impact on ColIV organization suggests that MMP1’s effects are indirect, perhaps by proteolytically regulating an unknown factor. Through this or another mechanism, PC-produced MMP1 fashions a BM framework whose specific texture drives appropriate elongation of the entire organ, enhancing reproductive fitness for the organism (Ku, 2023).
Current data do not allow us to define the specific mechanical property of the BM that MMP1 activity promotes. AFM of follicle BM has demonstrated that apparent elastic modulus assessed by probe indentation negatively correlates with the axis of increased elongation during growth and with sites where tissue rupture is most likely to occur under osmotic pressure (Ku, 2023).
Yet, this stiffness measured perpendicular to the BM plane seems unlikely to be the only property associated with the ability of the matrix to accommodate organ expansion. The striking increase in aspect ratio of MMP1-overexpressing follicles during osmotic swelling hints that MMP1 promotes enhanced amenability to tissue expansion without rupture. On the other hand, this study found that MMP1 also promotes the anterior narrowing that makes stage 8 follicles distinctly "pointy," both during development and in the osmotic swelling assay. This contribution seems inconsistent with a simple role for MMP1 in increasing BM pliability, and it highlights the knowledge gap between specific spatial organizations of ECM and their observed biomechanical properties (Ku, 2023).
These findings point to an unappreciated role of the two anterior PCs as specialized regulators of tissue mechanics. Live imaging demonstrates that these PCs are highly contractile, repeatedly tugging on the follicle BM, in contrast to the non-contractile neighboring epithelium (in which mechanosensitive vinculin plays no apparent role). Moreover, PC contractility increases with distant elevation of BM ColIV levels, consistent with a model in which PCs use focal adhesion sensing to assess the tissue rigidity landscape. That both static and migrating cells can sample substrate stiffness via cellular contractility is well-established (Ku, 2023).
Moreover, in several cancer cell lines, ECM composition has been found to regulate MMP activity, which may promote tumor cell invasion during pathology. This work identifies a physiological role for this signaling axis in sensing the global mechanical landscape during normal development, and then using the information to non-autonomously regulate BM patterning. It is interesting to note that the BM patterning role of PCs, mediated by MMP1 production, works in parallel to their Upd-mediated cell fate patterning role; indeed, ectopic PCs induced in the follicle epithelium are also contractile, produce MMP1, and promote ectopic pole-like bursting in the swelling assay. Such sufficiency as well as necessity data underscore how a few such specialized cells can be critical architects that shape the tissue mechanical environment (Ku, 2023).
As discussed above, although this study provides evidence that PCs can sense distant as well as local changes in BM properties, the mechanism by which they do so remains unknown. The data also do not reveal the direct effector that alters BM texture and mechanics in response to MMP1 activity provided by the PCs. Finally, while the swelling assay provides valuable information about tissue material properties, BM stiffness was not directly measured in this study, using AFM or related techniques. A full understanding of how the follicle uses the mechanisms described to ensure appropriate egg shape will be the subject of future work (Ku, 2023).
In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, the expression of Snail family transcription factor Escargot (Esg) was detected and further validated. A similar relationship was ascertained between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, these results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity (Chatterjee, 2023).
Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, this study conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance (Cho, 2018).
Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. This stufy reports identification and functional characterization of another member of the same protein family, CG3838/Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl(-) ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with the phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline (Shukla, 2018).
Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, this study fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, this study tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, it was found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. These results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal's true nutritional status (Alves, 2022).
The Drosophila ovary serves as a
model for pioneering studies of stem cell niches, with defined cell
types and signaling pathways supporting both germline and somatic stem
cells. The establishment of the niche units begins during larval stages
with the formation of terminal filament-cap structures; however, the
genetics underlying their development remains largely unknown. This
study shows that the transcription factor Lmx1a is required for
ovary morphogenesis. Lmx1a is expressed in early ovarian somatic
lineages and becomes progressively restricted to terminal filaments and
cap cells. Lmx1a is required for the formation of terminal filaments,
during the larval-pupal transition. Finally, the data demonstrate that
Lmx1a functions genetically downstream of Bric-a-Brac, and is crucial for the
expression of key components of several conserved pathways essential to
ovarian stem cell niche development. Importantly, expression of chicken
Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating
functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary (Allbee, 2018).
Drosophila ovaries are composed of about 15-20 functional units called ovarioles that produce eggs. In each ovariole, somatic and germline stem cells are maintained by a group of specialized cells that form the niche. These niches are composed of terminal filaments (TFs) and cap cells, which provide signals required for the maintenance and function of the stem cell populations in adult ovaries, including BMPs and Hh. Although the origin of the somatic lineage in the female gonad has been established, the genetic factors underlying the morphogenetic processes required for the specification and establishment of TF-cap structures are largely unknown. Ovarian somatic cells originate from three mesoderm cell clusters on each side of the embryo. During the mid-larval third instar (ML3), TF cells start to specify, expressing the TF marker Engrailed (En). By late third larval instar (LL3), and continuing in white pre-pupae (WPP), TFs form well-organized stacks and promote the proliferation and migration of muscle precursors that will develop into the muscular sheath and delimit individual ovarioles. This process ensures that a single TF stack and a defined population of germline and somatic cells are incorporated in each unit (Allbee, 2018).
The number of ovarioles and the developmental timing of the niche have been shown to be controlled by several signaling pathways, including Insulin, Hippo, Notch, Activin and Ecdysone pathways. In addition, the two transcription factors Bab1 and Bab2, encoded by the bric-á-brac (bab) locus, are expressed in TFs and are essential for the formation of these structures. Other BTB transcription factors, Pipsqueak, Trithorax-like and Batman (Lola like), also control TFs and ovariole numbers. Finally, loss of the Engrailed transcription factor causes abnormal TF cell stacking. Although these signaling pathways and transcriptional regulators are essential for development of stem cell niches, the early genetic events coordinating TF-cap cell specification, formation and function remain largely unknown (Allbee, 2018).
This study describes the expression and function of the LIM-homeodomain transcription factor Lmx1a in developing Drosophila ovaries. LIM-homeodomain proteins have essential roles during tissue patterning and cell differentiation in metazoans, from nematodes to vertebrates. Mammalian Lmx1a and Lmx1b are pleiotropic regulators of cell differentiation and tissue development in many organs, as shown by the defects and syndromes caused by loss-of-function mutations in these genes (Doucet-Beaupré, 2015). For instance, haploinsufficiency of Lmx1b causes nail-patella syndrome (NPS), a disorder affecting dorsal limb structures, kidneys, anterior eye components and the nervous system. Studies in genetic mouse models have confirmed a regulatory function of Lmx1b in the development of these organs and the behavior of associated cell types, including serotonergic neurons, podocytes and several eye tissues. Similarly, studies of the mouse Lmx1a factor have revealed diverse developmental functions, including brain patterning and cell fate decision, dopaminergic neuron differentiation and insulin expression. Finally, previous work has implicated Lmx1a and Lmx1b in a variety of other pathologies, including non-NPS renal disease, Parkinson's disease, and various forms of cancer, including ovarian epithelial carcinoma, highlighting the complexity of Lmx1a/b biology, as well as the urgent need to understand the function of these factors better (Allbee, 2018 and references therein).
Two LIM-homeodomain proteins of the LMX subgroup are encoded in the Drosophila genome: Lmx1a/CG32105 and Lmx1b/CG4328. Lmx1a is expressed in the LL3 eye imaginal disc and its overexpression in this tissue causes eye defects (Roignant, 2010; Wang, 2016). However, the molecular, cell biological and developmental functions of this transcription factor have yet to be described. This study shows that Lmx1a is expressed in somatic lineages in the developing ovary. Its expression becomes restricted to TFs and cap cells by LL3/P0 (freshly pupariated) and is maintained in the adult stem cell niche. Analyzing a CRISPR-generated Lmx1a knockout allele as well as cell type-specific and stage-specific knockdown of Lmx1a, this study has determined that Lmx1a is required for ovary development specifically in the TF-cap cell niche at the time at which it forms. Transcriptional profiling of developing ovaries was performed, and Lmx1a was placed downstream of Bab1/2 in the specification of TF cells. Without Lmx1a, several components of signaling pathways crucial to the forming niche are not properly expressed, including Hh, the transcription factors Sox100B, Engrailed and Invected, and confirmed that these genes are required in the Lmx1a lineage. Strikingly, expression of a chicken ortholog of Lmx1a in forming TF-cap cells is sufficient to rescue the Lmx1a null phenotype. It is anticipated that these results will further elucidate the genetic and cell biological mechanisms underlying the establishment of the Drosophila ovary stem cell niche and provide insight into the role of LIM-HD factors in tissue development and patterning, homeostasis and disease (Allbee, 2018).
Despite longstanding interest in the Drosophila ovary as a model for the adult stem cell niche, the genetic mechanisms underlying the initial establishment and development of the niche remain largely unexplored. To date, only two transcription factor loci, bric-á-brac and engrailed/invected, have been identified as required for the development or proper stacking of TFs, and their function remains largely unknown. This study has characterized the function of Lmx1a, a highly conserved LIM homeobox transcription factor, in the developing Drosophila ovary. By generating an Lmx1a knockout line as well as taking advantage of a novel collection of Gal4 drivers that produce spatially restricted developing ovary expression patterns, Lmx1a was shown to be required in TF-cap structures at the time at which they form and initiate ovarian stem cell niche establishment and ovariole morphogenesis. It remains unclear whether Lmx1a plays a role in TFs, cap cells, or both. The data also suggest that Lmx1a expression is maintained in the adult niche. Further studies will be required to determine whether it is essential for the function of the adult niche, for example by controlling the expression of self-renewal factors (Allbee, 2018).
Using RNAseq, a novel list of genes enriched in LL3/P0 ovaries was generated, and from this list several pathways have been identified that are potentially affected by the loss of Lmx1a. Within this list are several transcription factors and components of signaling pathways necessary for ovarian niche development, function and maintenance, including Hh, FGF, Notch, Engrailed and Sox100B. Indeed, RNAi-mediated knockdown of several of these transcripts within the Lmx1a-Gal4 lineage results in ovary developmental defects and reduced fertility. It is therefore proposed that Lmx1a controls many aspects of the development of this tissue, including proliferation, migration or cell shape changes, by regulating a series of developmental factors and signal pathways. It is important to note that this study does not allow distinguishing between a direct transcriptional regulation of these genes by Lmx1a and an indirect effect of an impaired development of the TF-cap structure in Lmx1a mutants. The data analyzed, in young adult females, the consequence of manipulating Hh, Sox100B, Engrailed and Invected in the Lmx1a lineage. Investigating the function of these genes, specifically during the LL3-P0 transition or later during metamorphosis or adulthood, will allow testing of whether their genetic requirement and expression are compatible with a direct regulation by Lmx1a and to understand better the sequence of events required for normal ovarian morphogenesis (Allbee, 2018).
Interestingly, Engrailed has been previously suggested to be required for terminal filament (TF) stacking based on clonal analysis of TF cells homozygous mutant for both Engrailed and Invected (Bolívar, 2006). The current work suggests that the stacking defects caused by these mutations lead to a mild developmental phenotype in young adults. However, consistent with other work revealing a crucial role for Engrailed in the adult niche, this study also shows that persistent knockdown of Engrailed/Invected well into adulthood leads to an eventual loss of ovaries. It is speculated that the progressiveness of this defect is the consequence of a continuous impairment of adult niche function that worsens a mild initial developmental phenotype (Allbee, 2018).
This study investigated where Lmx1a functions in relation to the transcription factors Bab1 and Bab2. In support of earlier work, this study found that both bab1 and bab2 are required for ovary morphogenesis. In addition, a strong reduction os shown in Lmx1a mRNA expression in the ovaries of LL3/P0 bab1 homozygous mutant animals, placing Bab1 genetically and/or temporally upstream of Lmx1a. It is worth noting that, although reduced, the expression of Lmx1a is not eliminated in Bab1 mutants. It is speculated that Bab2 activity may be maintaining residual Lmx1a expression in this background.Heterozygosity for Bab1 was found to be sufficient to enhance the ovary developmental defects caused by RNAi-mediated knockdown of the Lmx1a-dependent genes tested here, consistent with a model in which the bric-ì-brac locus lies genetically upstream of Lmx1a (Allbee, 2018).
Finally, this study found that the Lmx1a null homozygous phenotype can be significantly rescued by the expression of chicken Lmx1b, in terms of TF-cap formation, sheath formation and fertility. This reveals striking conservation between the Lmx1a/b factors across the animal kingdom. Even more striking is the conserved relationship between Lmx1a/b and Engrailed. For example, Lmx1b is required for Engrailed expression during both the establishment of the isthmic organizer in the developing mouse midbrain, and the specification and differentiation of dopaminergic neurons. Additionally, simultaneous lentiviral-mediated expression of Lmx1b and Engrailed has been shown to aid in the differentiation of dopaminergic neurons in vitro. Altogether, these observations point to a likely conserved Lmx1a/b-Engrailed transcriptional module involved in tissue patterning and cell differentiation, across different developing tissues, cell types and species. It is therefore anticipated that studies on the function of Lmx1a in the Drosophila ovary will lead to a better understanding of the developmental function of Lmx1a in animals ranging from invertebrates to mammals. This study also indicates that Drosophila represents a valuable model in which to investigate the mechanisms underlying complex diseases caused by a dysfunction of Lmx1a/b, such as nail-patella syndrome, ovarian carcinoma and Parkinson's disease (Allbee, 2018).
The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). This study used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. A null allele of the forked gene was generated using the CRISPR-Cas9 system, and forked was found not to be required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, it was demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal MT-organizing centers. These results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity (Baskar, 2019).
The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development (Durdevic, 2018).
This study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. This study thus demonstrates that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposon-mediated mutagenic effects (Durdevic, 2018).
The finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis, and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos. Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling. By tracing transposon protein and RNA levels and localization from the mother to the early embryos, it is suggested that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. This study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. This study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails (Durdevic, 2018).
A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis. It is possible that the absence of pole plasm in vas mutants results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future (Durdevic, 2018).
Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade. In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification. Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner, it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage. The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation (Durdevic, 2018).
Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. Previous work has shown that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, the gene was named anion exchanger 2 (ae2). This study generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, it was found that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and these studies provide new genetic tools to investigate the mechanisms by which this occurs (Benitez, 2019).
This study shows that the VRK-1 kinase BALL is required for self-renewal of germline stem cells in Drosophila, including the symmetrically amplifying PGCs of larvae and both male and female GSCs. These stem cells are actively maintained undifferentiated and they require BMP signalling for self-renewal that emanates from their cellular niche environments. In ball2 mutant female GSCs, where the requirement of BMP signalling for self-renewal is most pronounced, known targets of BMP signalling are regulated as in wild type GSCs. This indicates that BALL participates neither in the transmission nor the regulation of BMP signalling, and that it is needed to maintain stem cell character in a cell autonomous manner, irrespective of the tissue-specific maintenance signals that emanate from the niches (Herzig, 2014).
The loss of self-renewing stem cells could be caused by the induction of ectopic differentiation in these cells. The differentiation pathway was blocked in ball mutant female GSCs by removing also the central differentiation factor BAM. The results suggest that ball mutant GSCs are not eliminated from the stem cell niche because they initiate germline differentiation but that the GSCs differentiate because they lost the capacity for self-renewal (Herzig, 2014).
It is unclear by which mechanism BALL mediates the ability for GSC self-renewal. In ovaries, GSCs and FSCs undergo a regular turnover and are continuously replaced in the niche either by their own daughter cells or by symmetric divisions of the neighbouring stem cells. The replacement of GSCs involves competition between stem cells. Cells lacking BAM for instance, successfully displace less competitive wild type stem cells in the niche. However, if BALL is additionally removed from bam mutant cells, they appear to loose their competitive advantage. The molecular basis of stem cell competiveness is still poorly understood. However, it has been shown that overexpression of the Drosophila dMyc transcription factor diminutive enhances the competitiveness of GSCs and causes significantly enlarged nucleoli and increased rRNA expression in epithelial cells. These observations suggest a correlation between ribosome biogenesis and GSC competitiveness. Downregulation of ribosome biogenesis appears in fact to be directly required for germ cell differentiation, since BAM activates the Mei-P26 protein which downregulates the expression of dMyc. Furthermore, when overexpressed from a transgene, dMyc abrogates the tumour growth phenotype and the size increase of nucleoli in bam mutant cells. Additional support for the proposal that increased ribosome biogenesis in stem cells is crucial for their competitiveness and for maintaining their undifferentiated state derives from studies on wicked. Wicked is an essential component of the U3 snoRNP pre-rRNA processing, which is required to maintain the self-renewal of GSC. The findings that BALL is enriched in stem cell nucleoli and required for the structural integrity of nucleoli in tumourous GSCs provide a plausible link between ribosome biogenesis and BALL-dependent competitiveness of GSCs (Herzig, 2014).
Once displaced from the niche, ball2 mutant female GSCs differentiate according to their germline fate with only minor defects. This study did not address whether the differentiation of ball mutant cells is fully completed like in the respective wild type lineages in systems other than the adult female germline, but it was found, irrespective of the system examined, that BALL is not strictly required as proliferation factor. Especially the analysis of dividing follicle cells showed that BALL is not a cell cycle regulator. With two remarkable exceptions, BALL is also not essential for cellular survival. These exceptions, i.e., ball mutant PGCs at late larval stages and ball2 bamΔ86 double mutant germline cells outside the ovarian niche, represent conditions in which differentiation is either not supported by the tissue or not possible due to the lack of a differentiation factor. Therefore, it is tempting to speculate that BALL becomes only essential for cellular survival, when the ball mutant stem cells are unable to 'escape' from self-renewal into differentiation. Although ball clearly has multiple functions, e.g., oocyte chromatin organization or modulation of female PGC proliferation rate, the common defect observed in all systems examined so far is a failure in maintaining pools of undifferentiated cells. Since BALL is not an essential proliferation factor, these data suggest that also the additional defects in ball mutant larvae, i.e., lacking imaginal discs and degenerate brains, could be due to premature loss of undifferentiated progenitor or stem cells. Analysis of these systems will eventually show whether BALL is broadly required to maintain the undifferentiated state of cells during development (Herzig, 2014).
Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. This study reports that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade β-catenin. Disruption of β-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and β-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration (Lin, 2020).
Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. This study demonstrates that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, it was shown that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism (Simonet, 2020).
Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. This study shows that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. These results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin (DeLuca, 2020).
The work described here shows that the Drosophila female germline has multiple advantages for studying the developmental regulation of chromatin silencing both before and during differentiation. Female GSCs continuously divide to produce new undifferentiated progenitors, which expand and differentiate into nurse cells or oocytes, generating large amounts of a much simpler tissue than a developing embryo. Additionally, an inducible reporter assay compatible with the female germline was developed that sensitively responds to developmental changes in local chromatin repression in individual cells. In contrast to RNAseq, which measures steady state RNA levels, or ChIPseq, which correlates chromatin epitopes with their perceived function on gene expression, the reporters directly test how local chromatin influences the inducibility of surrounding genes, and are easily combined with tissue specific knockdowns to identify trans-acting factors contributing to reporter inducibility. Finally, a genetic engineering approach allows any construct (not just hsGFP) to be efficiently integrated into many pre-existing 'donor' sites, including those used previously with other reporters, or sites heavily silenced by repressive chromatin in differentiated cells. Although the number of different donor sites in certain types of chromatin is currently limited, new sites continue to be generated using CRISPR/Cas9 targeting and the method can ultimately be applied virtually anywhere in the genome (DeLuca, 2020).
Analysis of Polycomb repression with reporters, ChIP, and PcG-gene knockdowns provided numerous insights into how chromatin affects gene expression and female germline development in Drosophila. GSCs, the precursors of oocytes and nurse cells, contain a non-canonical, binary distribution of moderate H3K27me3 enrichment on all transcriptionally inactive loci and very low enrichment on active chromatin. A similar non-canonical H3K27me3 distribution was observed in early fly embryos, suggesting that noncanonical chromatin represents a 'ground state' for progenitors that will propagate future generations of undifferentiated germ cells or somatic cells that differentiate into specialized tissues. Such non-canonical chromatin was first identified in mouse oocytes and preimplantation embryos. If non-canonical H3K27me3 chromatin is a characteristic of undifferentiated, totipotent cells, what function might it confer to account for its conservation (DeLuca, 2020)?
Experiments confirmed previous workshowing that chromatin modified by PRC2 is essential for the Drosophila female germ cell cycle. Germline cysts lacking PRC2 are unable to stably generate oocytes, and E(z) GermLine-specific RNAi Knock Down (GLKD) nurse cells mis-express multiple genes and degenerate at about stage 5. In contrast, GSCs lacking PRC2 properly populate their niche, divide, and produce daughters that interact with female follicle cells and begin nurse cell differentiation. Removing PRC2 activity from GSCs did not generally increase the steady state abundance of genes or the inducibility of reporters in H3K27me3-enriched inactive or PcG domains. These results suggest that PRC2 and non-canonical chromatin lack vital functions in undifferentiated germline progenitors but are critical for repressing genes upon differentiation. However, a requirement for PRC2 or non-canonical chromatin under stress conditions or prolonged aging cannot be dismissed. For example, PRC2 could promote the long-term maintenance of female GSCs, similarly to how it maintains male germline progenitors in flies and mice (DeLuca, 2020).
Germline cysts and nurse cells are found in diverse animal species across the entire phylogenetic spectrum, but their function has been well studied mostly in insects such as Drosophila where they persist throughout most of oogenesis. While nurse cells have traditionally been considered germ cells rather than late-differentiating somatic cells, this study shows that that Drosophila nurse cells initiate Polycomb silencing and enrich PRC2 activity on a nearly identical collection of PcG domains as somatic cells. In more distant species, such as mice, nurse cells initially develop in a similar manner within germline cysts and contribute their cytoplasm to oocytes, but undergo programmed cell death before the vast majority of oocyte growth. Consequently, it remains an open question whether somatic differentiation plays a role in nurse cell function in mammals and many other groups (DeLuca, 2020).
The size and composition of oocyte cytoplasm are uniquely tailored to promote optimal fecundity and meet the demands of early development. In some species, including flies, nurse cells synthesize large amounts of specialized ooplasm to rapidly produce multitudes of large, pre-patterned embryos. In others, including mammals, oocytes more slowly synthesize the majority of ooplasm. Interestingly, both ooplasm synthesis strategies apparently require Polycomb silencing. However, the nurse cell-based strategy in flies primarily requires PRC2 but not PRC1 to silence hundreds of somatic genes, while the oocyte-based strategy in mice requires PRC1 but not PRC2 (DeLuca, 2020).
Different strategies of ooplasm synthesis may have evolved to be compatible with noncanonical germ cell chromatin. Staining experiments show that Drosophila oocytes maintain a widely distributed, non-canonical H3K27me3 distribution similar to pre-meiotic precursors or mouse oocytes, suggesting that non-canonical chromatin is conserved and maintained throughout the germ cell cycle. Similar to mouse oocytes, Drosophila spermatocytes also contain non-canonical chromatin and autonomously synthesize large amounts of cytoplasm by deploying PRC1 but not PRC2. Thus, three different types of germ cells are filled with large amounts of differentiated cytoplasm that requires Polycomb silencing for its synthesis, but nevertheless maintain a non-canonical, silencing-deficient PRC2 activity (DeLuca, 2020).
The conservation of undifferentiated, non-canonical chromatin despite a strong selection for Polycomb silencing during ooplasm synthesis argues that non-canonical chromatin must have a presently unappreciated fundamental purpose in germ cells. Noncanonical chromatin could regulate multigenerational processes like mutation, recombination, or transposition, that are not easily assayed in sterile individuals. Tests of these ideas will require a better understanding of how non-canonical chromatin is regulated and methods to disrupt non-canonical chromatin without disrupting other functions required for germline viability. Additionally, non-canonical chromatin could simply result from the silencing- incompetent PRC2 that was observed in progenitors (DeLuca, 2020).
Pcl was uncovered as both an inhibitor of PRC2 silencing and promoter of non-canonical chromatin in GSCs. PclGLKD dramatically altered the footprint of PRC2 activity in GSCs. PclGLKD favored H3K27me3 enrichment on PREs versus inactive domains, and increased the total amount of H3K27me1 by 13-fold and H3K27me2 by 1.4-fold and decreased the total amount of H3K27me3 by 1.8 fold. By binding DNA through its winged-helix domain, Pcl triples PRC2's residence time on chromatin and promotes higher states of H3K27 methylation in vitro. In GSCs, Pcl could simply change the result of each PRC2-chromatin binding event from H3K27me1 to me3. However, it is hard to imagine how an equivalent number of nucleosomes bearing a higher H3K27 methylation state could explain how Pcl inhibits silencing. Instead, it is proposed that Pcl inhibits silencing by reducing the number of PRC2-chomatin binding events per unit time by increasing the residence time of PRC2 on chromatin with each binding event. In this model, PclGLKD would not only convert many H3K27me3 nucleosomes into H3K27me1 nucleosomes, it would also convert many unmethylated nucleosomes into H3K27me1 nucleosomes. PclGLKD would more subtly affect H3K27me2 abundance because it simultaneously increases the number of PRC2-chromatin binding events while reducing the probability of each binding event leading to H3K27me2 versus me1 (DeLuca, 2020).
By reducing the number of PRC2 binding events, Pcl could increase the abundance of unmethylated H3K27 residues available for acetylation - a transcription promoting modification. In both flies and mammals, PRC2 transiently associates with chromatin to mono- and dimethylate H3K27 outside of traditional PcG domains, blocking H3K27 acetylation and antagonizing transcription. This study similarly found strong and widespread PRC2-dependent silencing in H3K27me1/2 enriched chromatin in nurse cells. Because inactive domain silencing was not affected by depletion of Pcl, Jarid2, or H3K27me3, it is proposed that core-PRC2, but not Pcl-PRC2 or H3K27me3, primarily silences inactive chromatin (DeLuca, 2020).
In GSCs, abundant Pcl could saturate PRC2, effectively depleting faster-sampling core-PRC2 complexes in favor of slower sampling Pcl-PRC2. In somatic embryonic cells, Pcl is present in a small fraction of PRC2 complexes. Compared to other fly tissues, Pcl mRNA is most abundant in the ovary, and within the ovary, Pcl protein is much more abundant in GSCs and nurse cell precursors than differentiated nurse cells and somatic cells. Within each differentiated germline cyst, Pcl mRNA is depleted from nurse cells and enriched in oocytes, suggesting that Pcl protein levels may be regulated by an mRNA transport mechanism induced in region 2 that also triggers the differentiation of oocytes from nurse cells (DeLuca, 2020).
Pcl, and a second PRC2-interacting protein, Scm, regulate the transition from noncanonical to canonical chromatin and initiate Polycomb repression. During nurse cell differentiation, it is proposed that Pcl depletion frees core-PRC2 to rapidly sample and silence inactive domains, while Scm (which is absent from the GSC) induction recruits high levels of PRC1 and PRC2 activity around PREs. ScmGLKD nurse cell chromatin retained a noncanonical H3K27me3 pattern characteristic of GSCs, as if differentiation at PcG domains had not occurred. In mice, Scm homologue, Scml2, similarly associates with PcG domains to recruit PRC1/2 and silence PcG targets during male germline development. However, unlike its fly orthologue in female GSCs, Scml2 is expressed in male germline precursors. This difference could explain why mammalian PGCs partially enrich PRC2 activity on CGIs while fly female GSCs do not enrich PRC2 on specific sites. While PcG domain-associated Scm is sufficient to enrich PRC2 activity above background levels found throughout inactive chromatin, a second PRC2 interacting protein, Pcl, is additionally required to promote full PRC2 and H3K27me3 enrichment on PcG domains. Because Scm oligomerizes and interacts with PRC2 in vitro, it could form an array of PRC2 binding sites anchored to PREs through Sfmbt. It is proposed that two cooperative interactions, PRC2 with PRE-tethered Scm, and Pcl with DNA, preferentially concentrate H3K27me3-generating Pcl-PRC2 versus H3K27me1/2-generating core-PRC2 on PcG domains (Figure 6D). H3K27me3 could then be further enriched by H3K27me3-induced allosteric PRC2 activation through the Esc subunit (DeLuca, 2020).
By promoting PRC1 and PRC2 concentration on PcG domains, Scm enhances silencing on PcG-localized reporters. While Scm-depleted nurse cells completed oogenesis, a subset of PcG domain-localized PcG including chinmo and the posterior Hox gene, Abd-b, escaped repression and were potentially loaded into embryos. Eggs derived from ScmGLKD nurse cells failed to hatch, and mis-expressed Abd-b in anterior segments following germ band elongation. This defect more closely resembled maternal plus zygotic than maternal-only Scm mutants, suggesting that ScmGLKD may deplete both maternal and zygotic Scm (DeLuca, 2020).
However, the additional possibility that mis-regulation of maternal Polycomb targets like chinmo contribute to the subtle embryonic defects observed in maternal-only Scm mutant clones cannot be excluded (DeLuca, 2020).
Further study of the Polycomb-mediated repression described in this study will help define the gene regulation program of Drosophila nurse cells and its contribution to oocyte growth. Additional characterization and perturbation of non-canonical chromatin throughout the germ cell cycle will yield further insights into its function in development. Finally, incorporating studies of other chromatin modifications, including H3K9me3-based repression, during germ cell development will contribute to a fuller understanding of how chromatin contributes to an immortal cell lineage (DeLuca, 2020).
How formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. This study shows that their functions are necessary specifically within precursor cells for TF formation during these stages. A new function was identified for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, the presence of Bab proteins in niche cells was shown to be necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary (Miscopein Saler, 2020).
Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. This study used a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, all phase portraits were enumerated of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, parameter regions were located that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches (Diegmiller, 2021).
The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1-domain (LEM-D) proteins that associate with the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). This study investigated the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only D-emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where D-emerin/Otefin function is essential. Germ cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to d-emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, these findings provide evidence for a prominent NL partnership between the LEM-D protein D-emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population (Duan, 2020).
The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear membrane. Comprising lamins and hundreds of associated proteins, the NL builds contacts with the genome to regulate transcription, replication and DNA repair. The NL also connects the nucleus with the cytoskeleton, facilitating transduction of regulatory information between cellular compartments. The composition of the NL is cell-type specific, providing a diverse platform for the integration of developmental regulatory signals. Changes in NL structure occur during physiological aging and disease, suggesting that maintenance of NL function is crucial for cellular health and longevity (Duan, 2020).
One prominent family of NL proteins are LEM domain (LEM-D) proteins, named after the founding human members: LAP2, emerin and MAN1. The defining feature of this conserved family is the LEM domain (LEM-D), an ~40 amino acid domain that directly interacts with the metazoan chromatin-binding protein Barrier-to-autointegration factor (BAF, sometimes referred to as BANF1). Purified human BAF directly binds double-stranded DNA, the A-type lamin and histones in vitro, suggesting that BAF also promotes chromatin-NL connections using non-LEM-D-dependent mechanisms. In dividing metazoan cells, regulated formation of complexes between LEM-D proteins, BAF and lamin controls mitotic spindle assembly and positioning, as well as the reformation of the nucleus. In non-dividing metazoan cells, LEM-D proteins and BAF cooperate to tether the genome to the nuclear periphery and form repressed chromatin. These properties highlight central connections between LEM-D proteins and BAF in NL function (Duan, 2020).
Studies in Drosophila melanogaster have begun to define the role of LEM-D proteins and BAF in development. Drosophila has three NL LEM-D proteins that bind BAF, including two emerin orthologues (Emerin/Otefin and Emerin2/Bocksbeutel) and MAN1. Each LEM-D protein is globally expressed during development. Even so, loss of individual NL LEM-D proteins causes different, non-overlapping defects in the several tissues, including the ovaries, testes, wings and the nervous system. These restricted mutant phenotypes reflect functional redundancy among the Drosophila LEM-D proteins, as loss of any two proteins is lethal. Strikingly, phenotypes of the emerin double mutants (otefin-/-; bocksbeutel-/-) phenocopy baf null mutants. Both baf and the emerin double mutants die before pupation, resulting from decreased mitosis and increased apoptosis of imaginal discs (Barton, 2014; Furukawa, 2003). In contrast, emerin/otefin; MAN1 or emerin2/bocksbeutel; MAN1 die during pupal development, without associated defects in mitosis or apoptosis. Together, genetic studies indicate that the Drosophila emerin orthologues and BAF are important partners (Duan, 2020).
This study extend investigations of the Drosophila NL LEM-D and BAF protein partnership. Using a CRISPR generated gfp-baf allele, this study confirmed that BAF is a globally expressed nuclear protein that shows strong enrichment at the NL in diploid cells. Strikingly, this NL enrichment largely depends upon one LEM-D protein, Emerin/Otefin. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where Emerin/Otefin function is essential. In germline stem cells (GSCs), loss of Emerin/Otefin causes a thickening of the NL and reorganization of heterochromatin. These structural nuclear defects are linked to activation of two kinases of the DNA damage response pathway: Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2). Although oogenesis in emerin/otefin mutants is rescued by loss of these DDR kinases, canonical triggers are not responsible for pathway activation. Instead, ATR and Chk2 activation is linked to defects in NL structure itself. Given the roles of BAF in mitotic nuclear envelope formation and repair , it was reasoned that checkpoint activation in emerin/otefin mutants might result from altered BAF function. This prediction was tested using germ cell-specific RNA interference (RNAi) to knockdown BAF. This study shows that BAF depletion disrupts NL structure, blocks differentiation and promotes GSC loss, mutant phenotypes that mirror Emerin/Otefin loss. Additionally, mutation of atr or chk2 partially restores germ cell differentiation in the baf mutant background, supporting the possibility that BAF depletion activates the NL checkpoint. Taken together, these findings suggest that Emerin/Otefin plays a dominant role in the enrichment of BAF to the NL and provide evidence that BAF functions with this prominent partner in the maintenance of an adult stem cell population (Duan, 2020).
This study extended in vivo studies of the BAF and LEM-D partnership. Capitalizing on a newly generated gfp-baf allele, this study shows that NL localization of BAF largely depends upon a single LEM-D protein, Emerin/Otefin. Loss of Emerin/Otefin is sufficient to disperse BAF in cells that express the A- and B-type lamins, Emerin2/Bocksbeutel and MAN1 in the NL. These data establish the in vivo existence of a prominent NL partnership between one LEM-D protein and BAF (Duan, 2020).
The basis for the unexpected reliance on Emerin/Otefin is unknown. One possibility is that LEM-Ds have different affinities for BAF. Pairwise alignment of amino acid residues within LEM-Ds shows the highest conservation between Drosophila emerin orthologues (70% similarity). Nonetheless, all LEM-Ds are strongly conserved in BAF-binding residues (42% identical, 67% similar). A second possibility is that the interaction of LEM-D proteins with BAF depends upon how a given LEM-D protein assembles into the NL network. Self-association of emerin influences both BAF and lamin binding. Finally, post-translational modifications (PTMs) of LEM-D proteins might impact BAF partnerships. As an example, O-GlcNAcylation modification of emerin affects BAF association, representing a regulated PTM that has the potential to alter NL function in response to nutrient availability. However, such signal-dependent PTMs are likely to be tissue specific, predicting a tissue-restricted, not global, effect on the NL enrichment of BAF. Further studies are needed to resolve the basis for the strong partnership between Emerin/Otefin and BAF (Duan, 2020).
BAF is essential for viability, with dying baf null larvae exhibiting a typical mitotic mutant phenotype that is associated with high levels of apoptosis. Several observations suggest that loss of NL BAF is not equivalent to complete loss of BAF. First, emerin/otefin null animals are viable, even though there is a global loss of NL BAF. Second, emerin/otefin null animals have lower levels of apoptosis in larval tissues than baf animals, without effects on the development of adult structures. Third, emerin/otefin mutant imaginal disc cells display an unchanged nuclear shape and chromatin architecture, whereas these cells are affected in baf mutants (Furukawa, 2003). Based on these data, it is suggested that BAF function at the NL during interphase is not essential. It is predicted that the essential BAF function relates to its contributions in mitosis and depends upon both Drosophila emerin orthologues, as these double mutant animals die with a mitotic mutant phenotype (Duan, 2020).
Effects of mislocalized BAF share features resulting from BAF overexpression in other systems. In emerin/otefin mutant germ cells, BAF dispersal contributes to the aggregation of heterochromatin. Defects in HP1a distribution have also been found in human cells overexpressing BAF or expressing a BAF mutant defective in interacting with NL components. Furthermore, several diseases affecting expression and processing of lamin A alter the distribution of BAF and resemble a BAF overexpression phenotype. Together, these findings support a model in which BAF contributes to the deleterious effects resulting from lamin or LEM-D mutations (Duan, 2020).
BAF is required for maintenance of Drosophila GSCs. Germ cell-specific BAF knockdown caused GSC loss, with remaining GSCs displaying a thickened and irregular NL structure, a phenotype shared with emerin/otefin mutants. These data support a model in which Emerin/Otefin and BAF function together to build NL structure in this cell type. Such a dependence on Emerin/Otefin for NL structure is consistent with limiting levels of the second Drosophila Emerin ortholog, Emerin2/Bocksbeutel (Barton, 2014). It is predicted that, in GSCs, the Emerin/Otefin and BAF might have a shared function in nuclear reformation at the end of mitosis (Duan, 2020).
Activation of the NL checkpoint is linked to NL deformation (Barton, 2018). Strikingly, baf mutant phenotypes are partially suppressed in atr/chk2; nos>bafRNAi animals, with double mutant ovaries showing increased germ cell survival and differentiation. Yet cell death remained in the double mutant backgrounds. Based on these observations, it is predicted that BAF loss in germ cells has multiple consequences. First, NL structure is affected. Second, loss of nuclear BAF might affect transcriptional networks required for GSC maintenance, suggested from studies showing BAF is an epigenetic regulator. Notably, the maintenance of mammalian stem cells also depends on BAF. Knockdown of BAF in either mouse or human embryonic stem cells promoted premature differentiation and reduced survival, phenotypes associated with an altered cell cycle. It remains possible that loss of Drosophila BAF in GSCs perturbs mitosis, which might induce apoptosis. Additional studies are needed to elucidate cell cycle contributions of BAF in GSCs (Duan, 2020).
These studies emphasize the important role of BAF within the NL network. Evidence is presented for consequences of BAF dispersal and loss during development, showing BAF dysfunction causes cell-type specific responses. Further definition of the developmental contributions of BAF will advance understanding of laminopathies, including the Nestor-Guillermo syndrome: a rare hereditary progeroid disorder caused by a missense mutation in BAF/BANF1 (Duan, 2020).
The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. This study reports that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, it was shown that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation (Hu, 2021).
Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. This study demonstrates that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. A known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division (Sardi, 2021).
The Drosophila female germline stem cell (GSC) is an excellent model to study niche-stem cell interaction because of its well-defined anatomy and abundant cellular markers. At the tip of each ovariole, two to three GSCs adhere to a cluster of niche cells, known as cap cells (CCs). A bone morphogenetic protein (BMP) ligand, Decapentaplegic (Dpp), is secreted by CCs and is an essential factor for GSC maintenance. Dpp binds to the serine-threonine kinase receptor Thickveins (Tkv) expressed on GSCs. Activated Tkv then phosphorylates Mothers against decapentaplegic (Mad) at its two C-terminal phosphorylation sites. Phosphorylated Mad (pMad) forms a heterodimer with Medea (Med), and the complex enters the nucleus and directly binds to the promoter of the differentiation factor bag of marbles (bam) to down-regulate its transcription. The down-regulation of bam is essential for GSC self-renewal (Sardi, 2021).
It has been hypothesized that the niche signaling rapidly decreases in one of the GSC daughters, precystoblast (preCB), as it is displaced away from the CC niche. Multiple studies have defined mechanisms for fine-tuning Dpp signal strength and range. However, it is still unclear how Mad, the immediate downstream molecule of Tkv, is initially regulated during GSC division (Sardi, 2021).
This study demonstrates that pMad rapidly reaches different levels in the dividing GSCs after mitosis but before the completion of cytokinesis. Its level in the nuclei of future GSCs remains high, while its level in the nuclei of preCBs decreases. Upon activation of the niche signal receptor Tkv kinase, its substrate, Mad, is phosphorylated near the plasma membrane and then travels throughout the cytoplasm and enters the nucleus. After mitosis, GSC takes several hours to complete cytokinesis, and the abscission occurs during DNA synthesis (S) phase. Since preCB shares its cytoplasm with GSC during this time, pMad can travel freely between the cytoplasm of two daughters. How can their nuclei have different levels of pMad (Sardi, 2021)?
This study shows that the previously identified Mad phosphatase Dullard (Dd) plays an essential role in the formation of the sharply different pMad levels in GSC and preCB. It was demonstrated that Dd interacts with Mad at the nuclear pores, where it may directly or indirectly dephosphorylate pMad. Dd itself does not exhibit asymmetric localization in GSC and preCB, but, as mathematical modeling indicates, the unbiased dephosphorylation by evenly distributed Dd combined with the phosphorylation of Mad biased toward the niche (due to local activation of Tkv on the niche side) is sufficient to explain the observed pMad asymmetry (Sardi, 2021).
In summary, these results provide a mechanism by which a self-renewal program is confined to the stem cells during asymmetric division (Sardi, 2021).
During asymmetric stem cell division, two daughter cells acquire different cell fates. The Drosophila ovarian niche differentially activates an GSC and a preCB because of the distinct position of these cells, the former adjacent to the niche and the latter displaced from the niche. What is the initial cause of the difference? While many factors have been identified that can amplify and/or ensure the already existent niche signal difference, it has been unknown how Mad, the immediate downstream molecule of the niche ligand, is initially regulated during GSC division (Sardi, 2021).
Mad is phosphorylated by active receptors at the side of GSC near the niche and then travels throughout the cytoplasm before entering the nucleus. The GSC-preCB pairs continue to share the cytoplasm for at least several hours after mitosis. Consistent with previous studies, this study observed that Mad diffuses throughout the cytoplasm of the GSC and preCB. This study found, however, that during this phase, the pMad levels in the nuclei of the GSC and preCB are already asymmetric (G1/S pMad asymmetry). It was determined that local activation of the kinase at the niche-GSC contact site is not sufficient for G1/S pMad asymmetry. Moreover, neither increasing the activity of kinase nor compromising the rate of Mad degradation affected G1/S pMad asymmetry. It was discovered that the Mad phosphatase, Dd, which dephosphorylates Mad at the nuclear pores in both GSC and preCB cells, is an essential factor in the formation of early asymmetric partitioning of pMad (Sardi, 2021).
To gain insight into the mechanisms responsible for the observed G1/S asymmetry, a mathematical model was formulated that included all major factors governing the spatiotemporal dynamics of pMad, and was constrained by the experimental data obtained in this study. The model suggests that a combination of the asymmetrically localized activated kinases, which reside at the site of contact between the GSC plasma membrane and the niche, together with sufficiently active phosphatase that is distributed symmetrically between nuclear envelopes of the GSC and preCB, is sufficient to explain the experimentally observed asymmetry of pMad levels in the GSC-preCB pairs (Sardi, 2021).
Analysis of the model revealed that for pMad asymmetry to occur, it is necessary that the positive and negative regulators of pMad (the kinases and phosphatases) be separated in space, as this brings about spatial gradients of pMad. However, this condition is not sufficient, as the gradients could be shallow. The modeling showed, furthermore, that the steepness of pMad gradients is exclusively determined by the interplay of two factors: pMad dephosphorylation that steepens the gradients and pMad diffusion in the cytoplasm that levels them out. Active regulation of the pMad diffusivity, which is determined by protein size and effective viscosity of the cytoplasm, is unlikely. This makes the phosphatase activity an essential factor determining pMad asymmetry. To a lesser degree, the asymmetry also depends on the permeability of nuclear pores to pMad export from the nucleus, since the dephosphorylation of pMad occurs inside the nucleus (Sardi, 2021).
In summary, this study identifies and explains a mechanism by which a stem cell can rapidly set up an initial asymmetry with respect to an extrinsic signal thus providing a conceptual framework for understanding the dynamics of niche-stem cell signaling (Sardi, 2021).
The niche controls stem cell self-renewal and progenitor differentiation for maintaining adult tissue homeostasis in various organisms. However, it remains unclear whether the niche is compartmentalized to control stem cell self-renewal and stepwise progeny differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells form a niche for controlling germline stem cell (GSC) progeny differentiation. This study has identified four IGS subpopulations, which form linearly arranged niche compartments for controlling GSC maintenance and multi-step progeny differentiation. Single-cell analysis of the adult ovary has identified four IGS subpopulations (IGS1-IGS4), the identities and cellular locations of which have been further confirmed by fluorescent in situ hybridization. IGS1 and IGS2 physically interact with GSCs and mitotic cysts to control GSC maintenance and cyst formation, respectively, whereas IGS3 and IGS4 physically interact with 16-cell cysts to regulate meiosis, oocyte development, and cyst morphological change. Finally, one follicle cell progenitor population has also been transcriptionally defined for facilitating future studies on follicle stem cell regulation. Therefore, this study has structurally revealed that the niche is organized into multiple compartments for orchestrating stepwise adult stem cell development and has also provided useful resources and tools for further functional characterization of the niche in the future (Tu, 2020).
Stem cells maintain adult tissue homeostasis through continuous self-renewal and generation of differentiated cells. Their self-renewal is shown to be controlled by the niche in the organisms ranging from Drosophila to mammals. Recently, stem cell progeny differentiation has also been proposed to be regulated by the niche in the Drosophila ovary. The differentiation process often consists of multiple developmental steps for generating one or several functional cell types. However, it remains largely unclear how the niche controls these differentiation steps at the cellular level (Tu, 2020).
The Drosophila ovary is an effective model for studying niche functions in regulating germline stem cell (GSC) self-renewal and differentiation. At the tip of the germarium, two or three GSCs contact cap cells anteriorly and inner germarial sheath cells (IGSs) (previously known as escort cells) laterally in region 1 (see scRNA-Seq Reveals Five IGS Subpopulations in the Drosophila Germarium). Immediate GSC progeny, cystoblasts (CBs), divide four times synchronously with incomplete cytokinesis to form interconnected mitotic cysts (MCs) (2-cell, 4-cell, and 8-cell cysts) and 16-cell cysts. IGS cells wrap around CBs and MCs in region 1 as well as 16-cell cysts in region 2a. Follicle cells begin to surround 16-cell cysts in region 2b and then form stage-1 egg chambers in region 3. Cap cells and anterior IGS cells form a niche for controlling GSC self-renewal through Dpp/BMP-mediated signaling and E-cadherin-mediated cell adhesion, whereas IGS cells form a niche for promoting differentiation partly by preventing bone morphogenetic protein (BMP) signaling. IGS cells utilize Hh, Wnt, epidermal growth factor receptor (EGFR), and Jak-Stat signaling to prevent BMP signaling in GSC progeny. Long IGS cellular processes are regulated by Hh and Rho-CDC42 small GTPase signaling, and cellular-process-mediated direct interactions are important for CB differentiation and cyst formation. Ecdysone signaling prevents IGS transformation into cap cells during development and is also needed in IGS cells for cyst formation, meiosis, and egg chamber formation. Therefore, two niches coordinately control GSC development in the Drosophila ovary (Tu, 2020).
While interacting with IGS cells, newly formed 16-cell cysts in region 2a undergo three important cellular events. First, those 16-cell cysts undergo two important meiotic events: chromosomal pairing and meiotic recombination. Second, both pro-oocytes form synaptonemal complexes initially, and only one of them becomes the oocyte. Third, 16-cell cysts change their round shape into a lens-like shape to ensure exactly one cyst is packaged into an egg chamber by follicle cells. Thus, it is tantalizing to speculate that IGS cells function as a niche for regulating these three important germ cell developmental events. Consistently, ecdysone signaling functions in IGS cells to promote meiotic entry and egg chamber formation possibly by maintaining IGS identity. This study used 10x genomics single-cell RNA-sequencing (scRNA-seq) to identify IGS subpopulations, which form linearly arranged niche compartments for orchestrating GSC self-renewal, CB differentiation, meiotic recombination, timely oocyte development, and cyst shape change. Therefore, it is proposed that the niche forms distinct subcompartments to control GSC maintenance and stepwise progeny differentiation in the Drosophila ovary (Tu, 2020).
Although IGS cells and their cellular processes form a niche for controlling GSC progeny differentiation, including CB differentiation, cyst formation, and the meiotic entry, it remains unclear whether IGS cells form distinct niche compartments that control different differentiation steps. This study used scRNA-seq to identify four IGS subpopulations, IGS1-IGS4, which are organized linearly along the germarium to interact with GSCs and their early progeny. Genetic manipulations were used to show that IGS1-IGS4 subpopulations form functionally separate compartments for controlling GSC maintenance, CB differentiation, meiotic recombination, timely oocyte specification, and cyst shape change. In addition, this study has molecularly defined the FCP population, which remain poorly studied because of the lack of suitable molecular markers. Therefore, this study has identified four IGS subpopulations that form subsequential niche compartments for controlling different steps of GSC progeny differentiation and has also molecularly defined the poorly studied FCP population, which opens the door for in-depth studies of IGS and FCP populations in the future (Tu, 2020).
Two recent scRNA-seq studies on the whole Drosophila ovary have identified different somatic cell types important for oogenesis. However, those two studies failed to identify the IGS subpopulations because of high complexities of somatic cell types and similarities of IGS cells. This study used GFP-based cell sorting and scRNA-seq to successfully identify four IGS subpopulations, IGS1-IGS4 and has further used gene-specific Gal4 lines and mRNA FISH to show their linear arrangement in the anterior germarium. IGS1 and IGS2 reside in region 1 to interact with GSCs and CBs/MCs, whereas IGS3 and IGS4 are located in region 2a to contact 16-cell cysts. IGS4 is the most posterior population directly contacting FSCs and FCPs and should be the niche for FSCs on the basis of the previous studies. Unfortunately, this study has not identified unique markers for IGS1-IGS3 subpopulations except IGS4, which specifically expresses bnb and GstS1. However, the combinatory gene expression patterns can still reliably separate IGS1-IGS3 subpopulations. For example, NetA is specifically expressed in IGS1 and IGS2, whereas croc and Hf are expressed in IGS1-IGS3. In the future, the split-Gal4 strategy will be used to generate IGS-subpopulation-specific Gal4 lines for further defining their molecular signatures and functions (Tu, 2020).
Previous studies have identified three general IGS markers, c587-Gal4, 13C06-Gal4, and PZ1444. This study has identified bin, vn, Nrt, mirr, dnc, CG7194, and CG42458 as new molecular markers for IGS and FCP cells. In addition, RNAi knockdown results have demonstrated that bin and vn maintain IGS cells and promote GSC progeny differentiation. In addition to germ-cell-mediated EGFR activation in IGS cells, this study has shown that IGS-expressing neuregulin-like EGFR Vn also contributes to EGFR signaling and thus IGS maintenance. Given that Wnt, Hh, and EGFR signaling are known to maintain IGS cells, it will be important to determine whether forkhead transcription factor Bin functions downstream of these pathways to maintain IGS cells. Transmembrane adhesion molecule Dpr17 is dynamically expressed in different IGS subpopulations in different germaria, suggesting that IGS cells could change their adhesive property dynamically. This could be a potentially exciting finding given that CBs, MCs, and 16-cell cysts have to move along the germarium by disengaging one IGS subpopulation and then engaging a new IGS subpopulation. This speculation needs future experimental confirmation. Therefore, this study has provided important insight into IGS subpopulations by uncovering new markers and functions and has also improved the ability to probe new IGS functions in regulating GSC development in the future (Tu, 2020).
Although recent studies have shown that IGS cells maintain GSCs and promote CB differentiation, none of these studies have attributed the functions to any specific IGS subpopulations. This study shows that secreted signaling molecules, NetA and Hf, are expressed in IGS1 and IGS2 to maintain GSCs. However, this study could not definitively demonstrate that only IGS1-expressing NetA and Hf contribute to GSC maintenance because of the lack of IGS1-specific Gal4 lines. Although it was shown that IGS cells are needed for CB differentiation and IGS2 directly contacts CBs/MCs, it was not possible directly demonstrate that IGS2 directly controls the differentiation of CBs and MCs into 16-cell cysts because of the lack of IGS2-specific Gal4 lines (Tu, 2020).
This study also shows that IGS3 and IGS4 control meiosis, oocyte specification, and cyst shape. IGS3 and IGS4 in region 2a extend their long cellular processes to wrap around newly formed H2AvD+ pre-meiotic and meiotic 16-cell cysts. These 16-cell cysts still have two pro-oocytes, but they only retain one oocyte and also undergo the round-to-lens shape change when surrounded by follicle cells in region 2b. Knocking down IGS4-expressing wun2 and GstS1 decreases the H2AvD+ 16-cell cysts in region 2a and increases the presence of round 16-cell cysts in region 2b, which fails to become lens-shaped, suggesting that IGS4 cells regulate meiotic recombination and cyst shape change. In addition, IGS-specific knockdown of GstS1, but not wun2, causes the presence of 2 pro-oocytes in stage 1 egg chambers. Consistently, H2126-mediated bin and smo knockdown in IGS4 can also decrease the H2AvD+ 16-cell cysts and increase the frequency of stage 1 egg chambers with two pro-oocytes, further supporting that IGS4 regulates meiosis and oocyte specification. However, the possibility that IGS3 cells might also regulate meiotic recombination and timely oocyte specification could not be completely ruled because of the lack of IGS3-specific Gal4 lines given that IGS3 expresses low amounts of wun2 and GstS1 and some of them also express H2126. In addition to one previous study showing that Ecdysone signaling in IGS cells regulates meiotic entry, this study has, for the first time, shown that IGS cells regulate meiotic recombination in 16-cell cysts. Given that the oocyte was previously thought to be determined entirely by the intrinsic mechanism, the differential RNA and protein transport caused by asymmetrically localized microtubules and fusomes, this is also the first time to show that IGS cells influence timely oocyte determination in Drosophila. Therefore, this study has provided important insight into IGS subpopulations in the regulation of GSC maintenance, cyst formation, meiotic recombination, timely oocyte determination, and cyst shape change, but the underlying signaling mechanisms await future investigation through generation of new genetic tools (Tu, 2020).
Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors (Asaoka, 2019).
How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development. In other species, germline progenitors are specified by inductive signals from surrounding tissues. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors, implying that this phenomenon is critical for separation of the germline from the soma (Asaoka, 2019).
In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage 3-4). In pole cells of blastoderm embryos (stage 4-5), the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos). Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting the function of positive transcriptional elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T). By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown (Asaoka, 2019).
Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3' UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3' UTR and recruiting Nos to the RNA/protein complex. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation. Furthermore, deletion of NREs causes hb translation in pole cells, suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-like-sequence-dependent manner. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) These findings led to a speculation that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3' UTRs (Asaoka, 2019).
This study reports that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3' UTR. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on these observations, it is proposed that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells (Asaoka, 2019).
Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells. However, this study found that expression of Impα2 protein was at background levels in pole cells. Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRE-like sequence) in its 3' UTR, it was assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, the expression of the Impα2 protein was first monitored in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos. Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells, these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells (Asaoka, 2019).
Whether this repression is mediated by the NRE-like sequence in the impα2 3' UTR was investigated. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells. By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells. Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied, as well as when impα2 ΔNRE mRNA was supplied. Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos, these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells (Asaoka, 2019).
The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides. The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5'-HWWDUGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex. Hence, it was asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, electrophoretic mobility shift assay (EMSA) was performed using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro. Nos and Pum together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT), whereas alteration of the NRE-like sequence (mut) abolished this interaction. These results demonstrate that Nos and Pum are able to interact with the impα2 3' UTR in an NRE-like sequence-dependent manner. The observations described above led to a conclusion that Nos, along with Pum, directly represses impα2 translation in pole cells (Asaoka, 2019).
Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS). It was predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, this study focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells. In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos. Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells. To determine whether this aberrant concentration of Ftz-F1 was caused by mis-expression of Impα2, this study expressed Impα2 ectopically in pole cells of normal embryos. To this end, impα2 mRNA in which the 3' UTR was replaced with the nos 3' UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3' UTR. The percentage of these embryos (impα2-nos3'UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells. These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1 (Asaoka, 2019).
Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells. Because Ftz-F1 is required for proper expression of ftz in the soma, it was asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells, but never expressed in pole cells. By contrast, in impα2-nos3'UTR embryos, ftz mRNA was rarely detectable in pole cells. It is assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, Impα2 was expressed in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3'UTR embryos); the frequency of ftz expression was drastically increased, compared to those of impα2-nos3'UTR embryos and the embryos lacking Pgc (pgc embryos). A similar situation was observed in embryos lacking both Pgc and Nos activities (pgc nos embryos). The percentage of embryos expressing ftz in pole cells was 82.8%, an increase relative to 35.8% in pgc embryos. Furthermore, ectopic ftz expression in pgc nos pole cells was suppressed by injecting double-stranded RNA (dsRNA) against impα2. Therefore, it is concluded that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc (Asaoka, 2019).
In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3'UTR embryos. Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3'UTR pole cells than pgc or impα2-nos3'UTR pole cells (S3 Fig). Expression of the sex-determination gene Sxl was examined in early pole cells, because Sxl is also repressed by nos in both male and female pole cells. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3'UTR, pgc, and pgc impα2-nos3'UTR embryos. By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3'UTR embryos than in impα2-nos3'UTR, and pgc embryos. These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/or Sxl in pole cells (Asaoka, 2019).
Nos is required in pole cells for mitotic quiescence, repression of apoptosis, and proper migration to embryonic gonads. Hence, it was asked whether mis-expression of Impα2 causes defects in these processes. First, using an antibody against a phosphorylated form of histone H3 (PH3), a marker of mitosis, whether pole cells enter mitosis in stage 7-9 embryos was investigated. Premature mitosis was detected in pole cells of nos embryos, as described previously, but never in pole cells of impα2-nos3'UTR or pgc impα2-nos3'UTR embryos. Second, using an antibody against cleaved Caspase-3, a marker of apoptosis, whether pole cells enter apoptosis in stage 10-16 embryos was investigated. Pole cells never expressed the apoptotic marker in impα2-nos3'UTR embryos, whereas in pgc impα2-nos3'UTR embryos, 20.4% of pole cells expressed the apoptotic marker. The latter was statistically indistinguishable from pgc pole cells, which have been reported to enter apoptosis. These data indicate that mis-expression of Impα2 does not affect apoptosis of pole cells even in the absence of pgc function. Last, whether mis-expression of Impα2 affects pole cell migration was investigated. The ability of pole cells to migrate properly into the embryonic gonads was never impaired in impα2-nos3'UTR embryos, and the percentage of pole cells entering the gonads in pgc impα2-nos3'UTR embryos was statistically indistinguishable from that of pgc pole cells, which has been reported to exhibit migration defect. These observations indicate that mis-expression of Impα2 does not induce premature mitosis, apoptosis, or mis-migration of pole cells. This can be partly explained by the facts that Cyclin B and hid mRNAs are the targets for Nos-dependent translational repression regulating mitosis and apoptosis in pole cells, respectively (Asaoka, 2019).
During the course of the experiments described above, it was observed that impα2-nos3'UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis. Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, the effect of impα2-nos3'UTR in pgc/+ background was examined. The percentage of dysgenic ovaries in pgc/+ impα2-nos3'UTR females derived from pgc/+ impα2-nos3'UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3'UTR females. In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present. Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3'UTR males derived from pgc impα2-nos3'UTR mothers mated with y w males were higher than those in pgc and impα2-nos3'UTR males. In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses, the data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, it cannot be tested whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed (Asaoka, 2019).
Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3. impα1/Kap-α1/CG8548 mRNA is not detectable in pole cells during early embryogenesis, and its protein product is ubiquitously expressed at a very low level throughout embryogenesis. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation, and production of Impα3 protein is upregulated during the blastoderm stage. Because Impα3 production was independent of maternal nos activity, it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become transcriptionally active during gastrulation, when Impα2 is undetectable in these pole cells. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes (Asaoka, 2019).
Depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone, probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, it is proposed that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators (Asaoka, 2019).
Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres. This study demonstrated that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, it is proposed that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi. Therefore, it is proposed that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development (Asaoka, 2019).
In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. This study demonstrates that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. It is concluded that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis (Kong, 2019).
The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. This study shows that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. This study has delineated a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest (Hampoelz, 2019).
Nuclear pore complexes (NPCs) bridge the nuclear envelope (NE) and mediate nucleocytoplasmic exchange. They are giant assemblies of about 110 MDa in animals with an elaborate structure and composition. About 30 different genes encode for NPC components, termed nucleoporins (Nups). Those are subclassified into scaffold Nups that assemble into a cylindrical architecture with a ~50 nm wide central channel; and intrinsically disordered phenylalanine-glycine rich FG-Nups that line this channel. Scaffold Nups assemble into the so-called Y and inner ring complexes that form the outer and inner rings, respectively (see The Nuclear Pore Complex as a Flexible and Dynamic Gate). FG-Nups (containing phenylalanine-glycine repeats) have the capacity to phase separate in vitro. In vivo, they establish a unique biophysical milieu within the central channel that is impermeable to inert molecules. FG-Nups transiently interact with nuclear transport receptors (NTRs, also called importins, exportins, or karyopherins) that form complexes with cargo and cross the permeability barrier. Transport directionality across the NE is ensured by the small GTPase Ran. RCC1, the RanGTP exchange factor (RanGEF) is chromatin associated and maintains a high RanGTP concentration in the nucleus. The Ran GTPase activating protein (RanGAP) binds to Nup358 (also called RanBP2) at the cytoplasmic face of the NPC and ensures high RanGDP levels in the cytosol. Although RanGTP displaces cargo from import complexes in the nucleoplasm, GTP hydrolysis disassembles export complexes once they arrive at the cytoplasmic face. Nup358 is absent from lower eukaryotes but essential in animals and involved in active nuclear transport, cell cycle progression, malignant transformation, and viral infection (Hampoelz, 2019).
NPC assembly is an intricate process. In multicellular organisms, two assembly pathways were described. First, the relatively rapid assembly of NPCs from pre-existing building blocks concomitantly with nuclear envelope (NE) reformation at the end of open mitosis is referred to as 'post-mitotic' assembly. This pathway is spatially directed to chromatin by the Nup Elys. Temporal control is provided by cell-cycle-dependent kinases and phosphatases. Second, interphase assembly is a relatively slow process that generates NPCs from scratch in order to double their number for the next mitosis. It proceeds from inside out through the NE and requires the active nuclear import of Nups. Here, Nup153 spatially directs the Y complex to the inner nuclear membrane (Vollmer, 2015; Hampoelz, 2019 and references therein).
Little is known about the early steps of NPC assembly that occur prior to membrane association. FG-Nups serve as a velcro for scaffold Nups. They, however, have a considerable aggregation propensity in isolation that has to be controlled during NPC biogenesis in vivo. Non-NPC-associated Nups are chaperoned by importin β. RanGTP dissociates importin β complexes and thereby releases Nups for interphase and post-mitotic NPC assembly. Likewise, RanGTP induces NPC assembly in vitro, but the in vivo relevance of this finding remains to be tested (Hampoelz, 2019).
In multicellular organisms, nuclear pores also reside in stacked membrane sheets of the endoplasmic reticulum (ER), termed annulate lamellae (AL). Those are particularly prominent in gametes and embryos of a multitude of species including Drosophila. In early fly embryos, AL insert into the NE in order to supply the rapidly growing nuclei with additional membranes and NPCs (Hampoelz, 2016). AL are therefore thought to be maternally provided NPC storage pools. How AL assemble in the absence of a nuclear compartment, which spatially coordinates the process in case of the two previously characterized pathways, remains elusive. This study has investigated AL-NPC biogenesis in vivo during Drosophila melanogaster oogenesis. AL-NPC biogenesis was shown to be vastly abundant during oogenesis. It depends on the condensation of Nups into compositionally different granules that are transported along microtubules (MTs) and regulated by Nup358 in concert with Ran and NTRs. This NPC biogenesis is mechanistically distinct from both canonical NPC assembly pathways and progresses away from chromatin. It is proposed that instead, Nup358 condensates fulfill the role of spatially directing NPC biogenesis, in the absence of a bona fide nuclear compartment (Hampoelz, 2019).
Little has been known about the biogenesis of AL and the spatial cues that allow NPC formation away from the nuclear compartment. This work addresses these questions during Drosophila oogenesis and suggests a third, non-canonical NPC assembly mechanism. Already in nurse cells, Nup358 condenses into large granules (see A Model for NPC Biogenesis beyond the Nuclear Compartment). Condensation might be fostered by local translation of nup358 transcripts that enrich at the surface of Nup358 granules in a translation dependent manner. In nurse cells, AL biogenesis is suppressed, and only limited NPC assembly is observed within Nup358 granules. This could be due to the available amount or configuration of ER membranes or because high cytoplasmic concentrations of RanGDP promote the formation of Importin-Nup complexes that prevent other Nups from condensation. Nup358 granules become assembly platforms for AL-NPCs once they travel through ring canals into the ooplasm. Scaffold and FG-Nups condense into oocyte specific granules, possibly facilitated by elevated levels of RanGTP that dissociates the respective Nups from Importin. In the oocyte, NPC precursor granule interactions are promoted by MT dependent granule dynamics. Upon interaction, granules transfer material and assemble Nups onto available ER membrane, ultimately leading to the formation of larger stacks with multiple membrane sheets. Those are inherited to the embryo where they supplement dividing nuclei with NPCs throughout early embryogenesis (Hampoelz, 2019).
The phase-separating properties of FG-Nups have been subject to extensive research in vitro. This study provides evidence for condensation of Nups in vivo. Several properties, namely the coalescence of Nup358 granules, the transfer of material between granules, the high molecular mobility within granules, and the contact shapes observed upon granule interactions, are hallmarks of biomolecular condensates. Such condensates are defined as 'non-membranous organelles'. Although AL inherently contain stacked membrane sheets, they retain at least some characteristics of a phase separated condensate such as a milieu that is distinct from the surrounding cytoplasm. These findings underline the importance of phase separation at membranes that was also observed in other biological systems (Hampoelz, 2019).
Several lines of evidence, namely 1,6-hexanediol treatment, depletion of BicD and embargoed, and the interference with the Ran nucleotide status suggest that condensation of Nups into NPC precursor granules is critical for AL biogenesis. It is further underlined by the fact that Colchicine treatment counteracts the RanQ69L phenotype by reducing the number of MT-promoted granule interactions. Condensation concentrates NPC constituents in a constrained volume within the large ooplasm and might prevent unspecific interactions of soluble Nups. MT dynamics enhances interactions of otherwise unmixed, compositionally heterogeneous NPC precursor condensates and is a prerequisite for NPC assembly from condensed granules. It also prevents unwanted fusion and relaxation of compositionally homogeneous condensates of the same type. Facilitated interactions of granules could be of particular importance in the highly viscous ooplasm, where cytoskeleton-induced streaming is critical for the efficient distribution of various components (Hampoelz, 2019).
The two canonical NPC assembly pathways rely on the stepwise and orchestrated assembly of soluble Nups or subcomplexes onto either anaphase chromatin or the NE surface during interphase, respectively. However, these spatial cues are absent in the ooplasm and alternative mechanisms to locally concentrate assembly modules must be important. It is believed that the condensation of Nups replaces the canonical cues, in line with previous work that had shown functions of natively unfolded FG-Nups to stabilize each other but also NPC scaffold components during yeast NPC assembly. Controlled interactions and material transfer between condensates might account for specific steps of assembly and even provide a certain order, although this concept remains to be further tested (Hampoelz, 2019).
The data strongly suggest Nup358 granules as assembly platforms, where NPCs are seeded onto ER membranes. Nup358 has no reported role in initiating the assembly process in both previously described pathways (Weberruss, 2016). On the contrary, during interphase Nup358 assembles rather late onto the NPC scaffold. Although such information is not available for post-mitotic assembly, its mitotic localization to kinetochores could indicate an early role for Nup358. Indeed, Nup358 is of structural importance for the pore scaffold, given that its loss destabilizes the outer Y complex at the cytoplasmic ring at NE-NPCs. It is thus conceivable that Nup358 could not only stabilize but also recruit scaffold components onto membranes. Post-mitotic and interphase NPC assembly are initiated by two distinct Nups, Elys and Nup153, respectively. Elys localizes the Y complex onto anaphase chromatin and is dispensable for interphase assembly but also for AL formation, given that its depletion induces AL. In contrast, Nup153 seeds NPCs during interphase assembly onto the inner nuclear membrane, and it has been suggested to have a similar role at the ER during AL biogenesis. This study, however, found that Nup153 is absent from AL in oocytes(Hampoelz, 2019).
Despite all molecular and conceptual differences, the common driving force for NPC biogenesis at and beyond the nucleus is Ran that coordinates the availability of Nups for assembly by dissociating them from NTRs. Nup358 binds RanGAP and directly links the NPC to Ran activity. At the NE this is eminent to ensure a sharp Ran gradient and thus efficient nucleocytoplasmic transport. This study shows that this interaction is preserved beyond nuclei, because RanGAP strongly enriches at Nup358 granules in a Nup358-dependent manner. One might speculate that within the RanGTP milieu of the ooplasm, RanGAP induces a local Ran gradient at Nup358 granules that drives NPC biogenesis; conceptually similar to the nuclear compartment for interphase or postmitotic NPC assembly (Figure 7C and 7C'). Thereby, the observed progressive dilution of Nup358 and RanGAP at Nup358 granules in the oocyte could be important to drive their progression into AL. It might be caused by ooplasmic RanGTP that favors complex formation between Crm1 and Nup358. Although this would be consistent with the observed embargoed gene silencing phenotype, the enhanced condensation of Nup358 upon global induction of RanGTP argues for an alternative interpretation: Nup358 functionally interacts with both, Importins and Crm1 under specific conditions. It is thus not clear how exactly it is being chaperoned. The phenotype observed under embargoed gene silencing conditions might be indirectly caused by disturbance of the spatial distribution of Ran and NTRs across nurse cells and oocytes, as indicated by the variety in phenotype across individuals. Yet it stresses the importance of spatially controlled Nup condensation to assemble AL-NPCs. In any scenario, NTR-mediated de-condensation of Nup358 and the consequent reduction of local RanGAP activity would regulate the progression of NPC biogenesis by determining the availability of soluble, 'assembly prone' Nups and the degree of mixing at granule interfaces (Hampoelz, 2019).
Various aspects of AL-NPC biogenesis are markedly different from both canonical NPC assembly pathways. During oogenesis, Nup condensation, local translation and MT dependent dynamics interplay with Ran activity in order to faithfully assemble AL in oocytes. They are inherited to the embryo where this pool of ready-made NPCs supplements nuclei during the rapid interphases of the blastoderm stage. Because AL are present in a plethora of species, similar mechanisms are likely to operate throughout animals (Hampoelz, 2019).
The orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. This study implemented variational optical flow analysis as a new approach to analyze the polarity of
microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, it was found that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, it was found that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead to a proposal that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, a possible mechanism is proposed for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds; to counteract the recruitment of Kinesin to microtubules (Drechsler, 2020).
The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. This study examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted an analysis of focal adhesion dynamics. The rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, it was found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion (Zhao, 2022).
Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, this study characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. It was further shown that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, evidence is provided that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, it was shown that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins (McCambridge, 2020).
To summarize, although Me31B's localization to the posterior of an oocyte is likely independent of Osk, Aub, and Dart5, its proper enrichment at the site may still rely on Aub. Together with a previous report that Me31B's localization pattern is not affected in vas and tud mutants, it is speculated that Me31B's localization in a developing oocyte may be independent of the Osk-Vas-Tud-Aub assembly pathway, but its proper enrichment at the posterior germ plasm may still depend on certain conserved germ plasm proteins like Aub. (McCambridge, 2020).
This speculation, together with earlier conclusions in this study, led to the proposal of a hypothetical model for Me31B localization and enrichment process in the germline cells (see Hypothetical model of Me31B localization and enrichment into germ plasm). In this model, Me31B and conserved germ plasm proteins, Osk-Vas-Tud-Aub, exist in distinct granules in the germ plasm, Osk-Vas-Tud-Aub in germ plasm granules and Me31B (possibly associated with Tral and Cup) in separate granules but in close proximity. Me31B granules use an Osk-Vas-Tud-Aub-independent mechanism to localize to the cortex and the posterior of a developing oocyte, then the posteriorly localized Me31B granules interact with the germ plasm granules, which is necessary for proper Me31B granule enrichment in the germ plasm. In the early embryos, Me31B proteins begin to degrade rapidly and become dispersed in the cytoplasm (McCambridge, 2020).
During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, protein-protein associations were examined of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed identification of 26 previously unknown associations, besides recovering several well characterized interactions. Interactions were odemtofoed between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans (Bansal, 2020).
Post-transcriptional regulation of gene expression plays an essential role during oocyte RNA-binding proteins (RBPs) mediate post-transcriptional gene regulation by determining molecular fates of target RNAs. In addition to RNA-binding domains, RBPs often have additional auxiliary domains. These auxiliary domains may function as effector domains for post-transcriptional gene regulation directly through enzymatic activity or indirectly by mediating protein-protein interaction. Identifying these effector domains and their molecular functions is critical to understand the roles of RBPs in post-transcriptional gene regulatory mechanism (Zhu, 2018).
MARF1 is an RBP consisting of one RNA-recognition motif (RRM) followed by several tandem LOTUS domains (Limkain, Oskar, and Tudor containing proteins 5 and 7. Also called OST-HTH). Previous studies showed that mouse MARF1 is required for completion of meiosis in oogenesis by reducing protein and mRNA levels of retrotransposons and a few endogenous genes. However, the molecular mechanism by which MARF1 regulates gene expression remains unclear (Zhu, 2018).
LOTUS domains are conserved in bacteria, fungi, plants, and animals. In animals, LOTUS domain proteins are expressed almost exclusively in the germline and are implicated in RNA regulation. In Drosophila, these LOTUS domain proteins include Oskar, Tejas (human TDRD5), Tapas (human TDRD7), and MARF1 (Meiosis Regulator And mRNA Stability Factor 1 = GC17018. Human MARF1). However, the molecular function of the conserved LOTUS domain is not fully understood (Zhu, 2018).
This work studied the biological and molecular functions of Drosophila MARF1 and its LOTUS domains. MARF1 is essential for proper oocyte maturation by regulating cyclin protein levels. When tethered to a reporter mRNA, MARF1 caused shortening of reporter mRNA poly-A tail and reduced reporter protein level. This activity was mediated by MARF1 LOTUS domain. Consistent with this finding, it was found that MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain. Furthermore, it was found that MARF1 binds cyclin A mRNA, shortens its poly-A tail, and reduces Cyclin A protein level during oocyte maturation. Thus, this study uncovered the biological and molecular functions of Drosophila MARF1 and defined its conserved LOTUS domains as a post-transcriptional effector domain to recruit the CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress translation of the mRNAs (Zhu, 2018).
MARF1 is expressed in late-stage oocytes and is required for proper oocyte maturation by regulating cyclin protein levels. MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain to shorten target mRNA poly-A tails and thus reducing cyclin protein levels without changing cyclin mRNA levels. Thus, MARF1 LOTUS domain is defined as a post-transcriptional effector domain that binds the CCR4-NOT deadenylase complex (Zhu, 2018).
Recent studies by others showed that the LOTUS domains of Drosophila Oskar, Tejas, and Tapas bind germline DEAD-box RNA helicase Vasa to stimulate Vasa ATPase and helicase activities. Crystallographic studies showed that the LOTUS domain of Oskar forms a homodimer and that each of the monomer subunits binds the C-terminal domain of the Vasa DEAD-box helicase core on the side opposite to the dimerization interface. In contrast, this study shows that the MARF1 LOTUS domain binds the CCR4-NOT deadenylase complex, but does not bind Vasa, Oskar, or another molecule of MARF1 (Zhu, 2018).
The LOTUS domains found in Oskar, Tejas, and Tapas, but not MARF1, have a C-terminal extension, which is required for interaction with Vasa. Hence the LOTUS domains are divided into two subclasses: (1) extended LOTUS (eLOTUS) domain that is present in Oskar, Tejas, and Tapas, has a C-terminal extension, and binds Vasa, and (2) minimal LOTUS (mLOTUS) domain that is present in MARF1, lacks a C-terminal extension, does not bind Vasa, and instead binds the CCR4-NOT deadenylase complex. Thus, although eLOTUS and mLOTUS domains share core sequence homology except for the C-terminal extension, they mediate distinct protein-protein interactions. Interestingly, eLOTUS proteins (Oskar, Tejas/TDRD5, Tapas/TDRD7) contain a single eLOTUS domain while mLOTUS proteins (MARF1) contain multiple tandem mLOTUS domains (Zhu, 2018).
This study also showed that MARF1 binds cyclin A mRNA. In MARF1null mutant late-stage oocytes, cyclin A mRNA poly-A tail is longer and Cyclin A protein level is increased, without change in the cyclin A mRNA level. The degradation rate of Cyclin A protein was not changed in MARF1null oocytes compared with control oocytes in vitro. These results indicate that in control late-stage oocytes, MARF1 post-transcriptionally regulates Cyclin A protein level by binding cyclin A mRNA, shortening cyclin A mRNA poly-A tail, and reducing Cyclin A protein level. In contrast, poly-A shortening of cyclin A mRNA is lost in MARF1null oocytes, resulting in an accumulation of Cyclin A protein. Cyclin A is the only protein found that is increased in its level in MARF1 mutant oocytes (Zhu, 2018).
Based on these findings, a model is proposed for MARF1 molecular function. The MARF1 RRM binds specific target mRNAs, such as cyclin A mRNA, and the MARF1 mLOTUS domains recruit the CCR4-NOT deadenylase complex (see Models for MARF1 function in oocytes). This results in shortening of target mRNA poly-A tail and reduction of protein level produced from target mRNAs. The multiple, tandem mLOTUS domain may recruit multiple CCR4-NOT deadenylase complexes per single MARF1 molecule and single target mRNA, enabling efficient poly-A shortening (Zhu, 2018).
Using this model, it is proposed that MARF1 reduces Cyclin A protein level in stages 12-14 oocytes. This regulated reduction of Cyclin A protein level leads to expression of Cyclin B and Cyclin B3 proteins. This cyclin proteins expresson profile leads to stabilization of CDK1 protein and phosphorylation of CDK1 Tyr15 residue. Increase CDK1 and phosphorylation of CDK1 Tyr15 residue result in phosphorylation of appropriate target proteins of CDK1 in stage 14 oocytes. Proper global protein phosphorylation profile allows germplasm localization of Vasa and Aub and normal yolk distribution in stage 14 oocytes. As stage 14 oocytes traverse through the oviduct, meiotic Metaphase I arrest is released to complete meiosis and produce normal eggs (Zhu, 2018).
Consequently, it is speculated that Cyclin A is the main and/or most upstream target of MARF1. Persisted Cyclin A protein level in MARF1 mutant late-stage oocytes arrest them in an abnormal state rather than proceeding to a normal stage 14 including decreased protein levels of Cyclin B and Cyclin B3. Dysregulation of the three cyclin proteins levels results in the decreased CDK1 protein level and the decreased Tyr15 phosphorylation of CDK1. Dysregulation of cyclins and CDK1 alters global phosphorylation pattern. The altered phosphorylation pattern results in the loss of germplasm localization of Vasa and Aub. These together cause meiotic failure and complete sterility in MARF1 mutants (Zhu, 2018).
MARF1 seems to target specific mRNAs for gene silencing in diverse species. This study identified cyclin A mRNA as a target of Drosophila MARF1. Mouse MARF1 reduces protein and mRNA levels of retrotransposons and a few endogenous genes such as PPP2CB, suggesting that they are targets of the post-transcriptional silencing by mouse MARF1. Knockdown of human MARF1 causes upregulation of IFl44L mRNA, suggesting that IFl44L mRNA a target of human MARF1. Post-transcriptional gene silencing of target mRNAs in fly, mouse, and human MARF1 suggests that mLOTUS-domain directed recruitment of the CCR4-NOT deadenylase complex may be a widely conserved mechanism (Zhu, 2018).
LOTUS domains are found not only in animals but also in bacteria, fungi, and plants. LOTUS domains found in bacteria, fungi, and plants are more similar to the animal mLOTUS domains since they lack the C-terminal extension found in the animal eLOTUS domains. This suggests that the mLOTUS domain may be more ancient than the eLOTUS domain. It will be interesting to investigate the functions of these LOTUS domains found in non-animals, particularly the function of bacterial LOTUS domains, since bacteria do not have a poly-A tail in their mRNAs or CCR4-NOT deadenylase complex (Zhu, 2018).
Kawaguchi, S., Ueki, M. and Kai, T. (2020). Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression. PLoS One 15(4): e0231114. PubMed ID: 32243476
Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. This study examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. These results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. It is proposed that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation (Kawaguchi, 2020).
Nos is an evolutionary conserved protein that play important roles in early embryogenesis, formation of primordial germ cells and maintenance of germline stem cells (GSCs). However, the function of Nos during late oogenesis has not been described yet. Nos protein expression reaches the maximum around stage 10 of oogenesis and immediately reduces thereafter. This suggests that Nos expression is tightly regulated during late oogenesis. The current results indicate that dMarf1 regulates Nos expression by inhibiting its translation in late oogenesis. In the absence of dMarf1 expression during early-to-mid oogenesis, Nos might repress cycB to prevent the premature release of meiotic arrest until stage 10 of oogenesis. These results suggest that dMarf1 may coordinate oocyte maturation during late oogenesis in Drosophila; moreover, dMarf1 is dominantly expressed after stage 10, and binds to nos mRNA to repress its translation and reduce its expression. Consequently, Nos expression almost disappears at stages 12-14. Reduced expression of Nos induces the release of cycB mRNA from repression and promotes CycB translation. Subsequently, CycB binds to Cdk1 to form the active MPF complex to promote meiosis. Therefore, dMarf1 plays an important role in the release of the second meiotic arrest to drive embryogenesis (Kawaguchi, 2020).
The MARF1 gene is evolutionarily conserved in animals; most proteins of the MARF1 family contain three major domains: NYN, OST (also known as LOTUS), and RRM. Although the ribonuclease activity of NYN is essential for MARF1 function in mouse, it is not required in Drosophila dMarf1. The OST domain is present in the proteins of several species ranging from bacteria to humans. Drosophila melanogaster has has four members of OST domain-containing proteins: dMarf1, Oskar (Osk), Tejas (Tej), and Tapas (Tap). All of these proteins except dMarf1 contain a single OST domain, and are predominantly expressed in germline cells. Structural and biochemical studies of Osk, Tej, and Tap OST domains have revealed the ability of this domain to bind to Vasa, an RNA helicase expressed exclusively in germline cells. Interestingly, the OST domain(s) of MARF1 family members is smaller than those of other proteins and does not bind to Vasa. Instead, a recent study reported that dMarf1 could bind to CCR4-Not deadenylase complex via the OST domain(s); however, the importance and cooperation between multiple OST domains remains elusive. In addition to these two domains, the MARF1 family members contain one or two RRM domains. This study showed that dMarf1 translationally repressed nos mRNA. nos may not be the only target of dMarf1; other mRNAs such as tra2 and abo can also be targeted by dMarf1, although the biological significance of these interactions remains unclear. Zhu has recently reported that dMarf1 can bind to cyclin A mRNA via its RRM domain (Zhu, 2018). However, RNA-IP analysis did not detect cyclin A in the dMarf1-bound mRNA fraction. Further studies of the molecular mechanism underlying the specificity of dMarf1 RNA binding will reveal the range of mRNA regulation during oocyte maturation (Kawaguchi, 2020).
The C-terminal region of the MARF1 family members is highly conserved among higher animals, except insects, despite not forming any secondary structure. The C-terminal region of human MARF1 has been shown to directly interact with the decapping complex component Ge-1; however, this interaction was not observed in Drosophila. Moreover, the C-terminal region of the MARF1 family members is conserved among different insect species, but different from that of higher animals, indicating that it may bind to a unique partner in insects. This hypothesis was supported by results showing that transgenic dMarf1 mutant lacking 47 C-terminal residues (ΔC47) could not rescue dMarf1KO321 mutant phenotype (Kawaguchi, 2020).
In addition to binding to the RNA decapping complex subunit, human MARF1 can localize to processing body (P-body), which is often related to translational repression and mRNA decay. Mouse MARF1 has also been shown to degrade target mRNA via its NYN domain, which is absent in Drosophila dMarf1. Transcriptome analysis of mouse MARF1 mutant oocytes revealed that 1,470 transcripts were upregulated in the steady state, whereas 103 transcripts were downregulated, indicating a global impact on RNA homeostasis in the mutant oocytes. By contrast, the expression of a few RNAs was downregulated and dp1 mRNA expression was upregulated in Drosophila dMarf1 mutant ovaries. These results suggest that mammalian MARF1 may regulate the global transcriptome predominantly by degradation, while dMarf1 represses translation of target proteins such as CycB and CycA by modulating Nos/Pumilio and the CCR4-NOT deadenylase complex, respectively (Kawaguchi, 2020).
In addition to nos mRNA, dMarf1 can bind to other mRNAs, including tra2 and abo. The mRNA expression of tra2 was not significantly affected in stage 14 dMarf1KO312 egg chambers, suggesting that dMarf1 post-transcriptionally regulates tra2 expression. abo is a negative regulator of histone gene expression and its expression is downregulated in mature oocytes to produce more histones. The expression of abo mRNA was upregulated by approximately three-fold in stage 14 dMarf1KO312 egg chambers. This may result in the overexpression of Abo protein in dMarf1 mutant oocytes, which in turn causes the downregulation of histone proteins that are required for embryogenesis (Kawaguchi, 2020).
The Ppp2cb gene encodes a protein phosphatase that is involved in cell cycle regulation. Ppp2cb has been previously reported as a major downstream effector of mouse MARF1. The high expression of Ppp2CB phosphatase in the MARF1 mutant ovaries of mouse can disrupt meiosis. However, the expression of mts, a Drosophila homolog of Ppp2cb, was not affected in dMarf1 mutant ovaries, suggesting that the signaling pathway for the activation of the M phase promoting factor, CycB/Cdk1, is not conserved between mouse and Drosophila. Although the direct activation of MPF in mouse MARF1 mutant oocytes by the inhibition of Ppp2cb rescued meiotic defect, embryogenesis of mutant oocytes was affected, suggesting that Ppp2cb may have additional functions in addition to MPF activation. Similarly, dMarf1KO312 ovaries exhibited not only meiotic defects, but also translationally downregulated some proteins required for embryogenesis, such as Dhd and Gnu. In conclusion, MARF1 may trigger oocyte maturation and coordinate multiple events during late oogenesis and fertilization (Kawaguchi, 2020).
Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. This study shows that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. Functional evidence is provided that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. These findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes (Carvalho-Santos, 2020).
Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases. This study reports that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size (Ahmed, 2020).
Steroidal sex hormones including oestrogen, progesterone and testosterone regulate the growth and physiology of reproductive organs during puberty, the oestrus cycle and pregnancy. Consequently, these hormones also promote tumorigenesis in the breast, uterus and prostate. Although sex-specific differences in physiology and disease predisposition extend to nearly all organs, the functions of sex-specific steroids in non-sex organs remain relatively poorly explored and controversial. Drosophila uses one major steroid hormone, 20-hydroxy-ecdysone (ecdysone, also known as 20HE) and its derivatives. Similar to vertebrate steroids, 20HE is synthesized by cytochrome P450 enzymes from cholesterol. The ecdysone receptor comprises a ligand-binding EcR subunit and a DNA-binding Usp subunit-orthologues of human farnesoid X and liver X receptors (FXR and LXR) and retinoid X receptor (RXR), respectively. In juvenile insects, 20HE regulates developmental transitions including moulting, metamorphosis and sexual maturation. In adult Drosophila, 20HE is made by the ovaries after mating, resulting in higher levels in females than in males. It acts in the adult nervous and reproductive systems and affects metabolism and lifespan, but a role in the gut has not been described (Ahmed, 2020).
Drosophila intestinal stem cells (ISCs) are more proliferative in females than in males, and females are more prone to age-dependent gut dysplasia and intestinal tumours. These sex-specific traits could be due to ISC-autonomous and/or systemic factors. Consistent with the former, stress-dependent ISC divisions, which are more frequent in females than in males, are reduced if the ISCs are masculinized by repressing the sex-determination genes sxl or tra2. Mated females support more ISC division than virgin flies, which suggests hormonal influences. Because mated females have higher titres of ecdysteroid than virgins or males, tests were performed to see whether 20HE might affect ISC proliferation. Indeed, feeding virgin females 5 mM 20HE strongly induced ISC divisions. This effect was independent of ISC sex identity, and also occurred in mated females and males. Using reporters of receptor activity, it was confirmed that exogenous 20HE promotes EcR-Usp signalling in midgut ISCs, transient progenitors known as enteroblasts (EBs) and differentiated absorptive enterocytes (ECs) (Ahmed, 2020).
Unlike stress caused by detergents, 20HE treatment induced two successive waves of ISC division. Using RNA interference (RNAi) under the control of conditional cell-type-specific Gal4 drivers, it was found that the first wave (at 6 h after 20HE feeding) required EcR only in ISCs, but that later divisions (at 16 h) also depended partially on EcR in EBs. Neither wave of division required EcR in ECs, enteroendocrine or neural cells. Isoform-specific tests revealed that EcR-A was much more important than EcR-B for the 20HE-induced division of ISCs. 20HE-induced divisions were reversible , which suggests a lack of toxicity. EcR activity was not induced by enteric infection, and EcR was dispensable for infection-induced gut regeneration, which indicates a distinct role for EcR in the gut. Loss of Usp, however, did block infection-induced ISC divisions, which suggests that Usp has EcR-independent functions (Ahmed, 2020).
It was next asked whether ISC activation by 20HE involves the Upd-Jak-Stat or Egfr-ERK signalling pathways, which are known to activate ISCs after stress. Six hours of 20HE feeding induced the Egfr ligands spi and krn and their activating protease rho, but not the upd2 or upd3 cytokines or Stat signalling. Exposure to 20HE for 16 h, however, moderately induced upd2, upd3 and Stat activity. The induction of upd2, upd3 and rho required EcR in ISCs and EBs (that is, 'progenitors'), although not in ECs. The Egfr effector ERK was also mildly activated by 16 h of 20HE exposure, mostly in progenitors but occasionally in ECs. ERK activation required upd2, which suggests a signalling relay. Notably, the induction of all of these targets (upd2, upd3, Socs36E, rho, spi and krn) by 20HE was suppressed by blocking ISC mitoses with RNAi molecules that target string (also known as stg or cdc25) or Egfr. This suggests that the observed increases in Jak-Stat and Egfr-ERK signalling are responses to epithelial stress from the early ISC divisions. In further tests, it was found that Upd2 from EBs and ECs contributed strongly to ISC divisions 16 h after 20HE feeding, but only weakly to the early divisions at 6 h. Egfr and Rho, however, were always required. It is concluded that ISC divisions are initially activated ISC-autonomously via EcR, and require Egfr and Rho, whereas later divisions depend in part on cytokines produced by EBs and ECs, perhaps in response to stress from the first mitoses. The relationship of EcR to Egfr signalling warrants further investigation (Ahmed, 2020).
Because mated females produce more ecdysone than virgins or males, whether 20HE might account for sex-specific differences in the gut was tested. Consistent with this, long-term exposure of males to 20HE phenocopied the female condition, increasing ISC mitoses, stress responsiveness, epithelial turnover and midgut size. Genetically feminizing the male ISCs did not give these effect, which suggests that 20HE acts independently of genetic sex determination. Forced expression of the ISC mitogen13 sSpi also failed to enlarge male midguts, which indicates that 20HE affects more than just the ISC mitotic rate. Long-term 20HE feeding also endowed ISCs in virgin females with proliferative characteristics similar to those seen after mating. By contrast, RNAi lines that antagonized 20HE signalling in ISCs and EBs decreased gut size in mated females and suppressed mitoses in response to detergent stress. Thus, sexually dimorphic proliferative traits of ISCs are determined in part by 20HE signalling (Ahmed, 2020).
Similar to human oestrogen and progesterone, ecdysone promotes behavioural and metabolic changes that enhance female reproduction. Dose-response assays showed that 1 mM 20HE fed to virgin females activated EcR targets and ISC mitoses to similar levels to mating. Hence, this study tested whether endogenous, mating-induced 20HE activates ISCs. Indeed, mating induced a large, transient increase in ISC division and enduring gut enlargement. This was independent of genetic sexual identity. As with exogenously fed 20HE, these effects initially required EcR only in ISCs, although EcR in EBs contributed later on. Similar to exogenous 20HE, mating also induced expression of upd2 and rho, which suggests that these are normal physiological responses (Ahmed, 2020).
To confirm the source of endogenous ecdysone, ovary-specific Gal4 drivers were used to express RNAi transgenes that target the ecdysone synthesis enzymes Dib or Spo. This suppressed mating-induced ISC divisions and midgut growth, both of which could be restored by exogenous 20HE. spo mutants also failed to resize the midgut after mating, confirming these results. To learn how the gut grows in mated females, the effects on cell size and number. Depleting EcR in ECs did not reduce EC size, but mating caused a large 20HE- and EcR-dependent increase in female ISC numbers was investigated. This expansion of the stem-cell pool could cause an increase in the total number of midgut cells. These results indicate that mating-dependent ISC division, ISC expansion and gut growth are driven by 20HE signalling from the ovaries to progenitor cells in the gut (Ahmed, 2020).
Gut growth after mating is expected to increase the absorption of nutrients by the intestine and the supply of nutrients to other organs. Because egg production is limited by nutrient availability to the ovaries, whether 20HE-dependent gut growth affected female fecundity was tested. When gut resizing was blocked by expressing EcR RNAi (EcRRNAi) in midgut ISCs, or in both ISCs and EBs, egg production was reduced by approximately 40%. This suggests that 20HE-dependent gut remodelling maximizes female reproductive fitness. However, it was also noticed that the Gal4 drivers were active in a small number of escort cells in the germarium of the ovary, which raises the possibility that these fecundity defects were due in part to a requirement for EcR in those cells (Ahmed, 2020).
A study of Drosophila juvenile hormone (JH), a sesquiterpenoid, came to conclusions similar to the current study-namely that JH promotes mating-dependent gut growth and fecundity in females. The relative roles of 20HE and JH were therefore invstigated. The JH receptors Gce and Met are essential for ISC divisions in response to not only the JH receptor agonist, methoprene, but also 20HE and infection. The mitogenic effects of methoprene were confirmed, but these were weaker than those of 20HE or mating. Furthermore, it was discovered that methoprene-stimulated divisions require 20HE, and that JH or methoprene could suppress ISC divisions driven by 20HE or other stimuli. Although these results indicate interaction between 20HE and JH, further work is required to determine their precise physiological relationship (Ahmed, 2020).
To better understand how ecdysone activates ISCs, two known EcR targets were tested: the transcription factor Broad, and the nuclear receptor Eip75B, a homologue of human PPARγ and REV-ERB17. Eip75B and broad (br) mRNA were induced in midguts by 20HE or mating, and progenitor-cell-specific depletion of either factor suppressed 20HE-induced mitoses. ISC clonal growth, however, required Eip75B but not br, highlighting that Eip75B is a more essential effector. Overexpression of Eip75B was sufficient to promote ISC division and gut epithelial turnover, whereas Eip75B loss impaired both ISC mitoses and maintenance. Progenitor-specific loss of Eip75B also blocked gut growth after mating, and compromised egg production, phenocopying the effects of EcR loss. Eip75B binds DNA to repress target genes, and also binds the nuclear receptor Hr3 to inhibit Hr3-mediated transcriptional activation. Consistent with this mechanism, overexpression of Eip75B or 20HE feeding suppressed an Hr3 activity reporter, and Hr3 overexpression suppressed ISC proliferation. Moreover, depletion of Hr3 counteracted losses in ISC proliferation caused by Eip75B depletion. Although these results indicate that Hr3 is a crucial Eip75B effector, Hr3 loss was not sufficient to activate ISC division, which indicates that Eip75B has other targets. Further tests revealed that Eip75B and Hr3 mediate 20HE-independent ISC responses to stress. Enteric infection strongly induced levels of Eip75B mRNA and suppressed Hr3 activity. Removing Eip75B or Broad from ISCs by mutation or by RNAi blocked infection-induced ISC mitoses, as did overexpression of Hr3. Eip75B was also required for ISC mitoses in response to the oxidative stress agent paraquat. Furthermore, evidence was ontained consistent with previous work that the action of Eip75B is modulated by haem (a Eip75B ligand) and nitric oxide. Functions for haem and nitric oxide in the fly gut are unknown, but potentially interesting. It is concluded that Eip75B, Broad and Hr3 integrate several inputs in addition to 20HE to control ISC proliferation (Ahmed, 2020).
As females age, they experience progressive gut dysplasia in which ISCs overproliferate and mis-differentiate, leading to high microbiota loads (dysbiosis), barrier breakdown and decreased lifespan. Age-dependent intestinal dysplasia is more pronounced in females than in males, and can be identified by increases in mitoses and mis-differentiated cells doubly positive for ISC and EC markers. Suppressing EcR, Usp or Eip75B in midgut progenitors significantly reduced both parameters of dysplasia in aged flies. Similarly, suppressing ecdysone synthesis enzymes (Dib, Spo) in the ovaries, or ubiquitously, also curtailed age-dependent gut dysplasia. This effect could be reversed by 20HE supplementation. These results indicate that age-dependent gut dysplasia is potentiated by ovary-derived ecdysone, explaining the sex bias of this condition (Ahmed, 2020).
Female Drosophila are known to be more susceptible than males to genetically induced ISC-derived tumours. This study found that ISC/EB-specific RNAi targeting Notch, a receptor required for EC differentiation, drove tumour induction in 100% of mated females but was far less tumorigenic in virgin females or males. Three results indicate that this tumour predisposition is modulated by 20HE. First, in contrast to mated females, virgins were extremely resistant to NotchRNAi-mediated tumorigenesis. Second, targeting 20HE signalling in ISCs with a dominant-negative EcR-A (EcRADN) inhibited tumour growth in mated females. Third, supplementing males or virgin females with 20HE increased tumour initiation and growth. It is surmised that the use of mating-dependent, ovary-derived 20HE to stimulate gut resizing comes at a cost: it predisposes females to gut dysplasia and tumorigenesis (Ahmed, 2020).
Gut dysplasia, tumorigenesis and egg production can all shorten lifespan, which suggests that the effects of ecdysone on the gut might adversely affect longevity. In fact, earlier reports showed that EcR mutants live longer, and proposed that reproduction can shorten lifespan by damaging the soma. The lifespan assays of this study, although subject to the same caveats as previous work, support this view: suppression of EcR in midgut progenitors extended lifespan in females but not males. In evolutionary terms, the disadvantage of a slightly shorter lifespan due to sex-specific hormonal signalling is probably insignificant relative to the reproductive fitness advantage conferred by increased egg production. This may be especially true in the wild, where gut dysplasia-dependent mortality is probably counteracted by nutrient deprivation (Ahmed, 2020).
Similarities in the reproductive biology of Drosophila and mammals suggest that these inter-organ relationships have relevance to human biology. The mitogenic effects of insect ecdysone parallel those of oestrogen and testosterone as drivers of breast, uterine and prostate growth and tumorigenesis. Yet how these steroids affect the human intestine remains poorly explored. Adaptive growth of the intestine is well documented in pregnant and lactating mammals, and might depend on oestrogen and/or progesterone. Laboratory tests with rodents and human cells, as well as some studies with human participants, have linked oestrogen, testosterone and their receptors to gastrointestinal cancers, but epidemiological studies provide conflicting evidence regarding this association. The contributions of sex steroids to intestinal physiology deserve more detailed study (Ahmed, 2020).
Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. This study reports a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction (Gnainsky, 2021).
The fate and proliferative capacity of stem cells have been shown to strongly depend on their metabolic state. Mitochondria are the powerhouses of the cell being responsible for energy production via oxidative phosphorylation (OxPhos) as well as for several other metabolic pathways. Mitochondrial activity strongly depends on their structural organization, with their size and shape being regulated by mitochondrial fusion and fission, a process known as mitochondrial dynamics. However, the significance of mitochondrial dynamics in the regulation of stem cell metabolism and fate remains elusive. This study characterized the role of mitochondria morphology in female germ stem cells (GSCs) and in their more differentiated lineage. Mitochondria are particularly important in the female GSC lineage. Not only do they provide these cells with their energy requirements to generate the oocyte but they are also the only mitochondria pool to be inherited by the offspring. The undifferentiated GSCs predominantly have fissed mitochondria, whereas more differentiated germ cells have more fused mitochondria. By reducing the levels of mitochondrial dynamics regulators, it was shown that both fused and fissed mitochondria are required for the maintenance of a stable GSC pool. Surprisingly, it was found that disrupting mitochondrial dynamics in the germline also strongly affects nurse cells morphology, impairing egg chamber development and female fertility. Interestingly, reducing the levels of key enzymes in the Tricarboxylic Acid Cycle (TCA), known to cause OxPhos reduction, also affects GSC number. This defect in GSC self-renewal capacity indicates that at least basal levels of TCA/OxPhos are required in GSCs. These findings show that mitochondrial dynamics is essential for female GSC maintenance and female fertility, and that mitochondria fusion and fission events are dynamically regulated during GSC differentiation, possibly to modulate their metabolic profile (Garcez, 2020).
Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. This study investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. The analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. It is proposed that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte protein composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis (McDonough-Goldstein, 2021a).
The zinc finger-associated domain (ZAD) is present in over 90 C2H2 zinc finger (ZNF) proteins. Despite their abundance, only a few ZAD-ZNF genes have been characterized to date. This study systematically analyzed the function of 68 ZAD-ZNF genes in Drosophila female germ cells by performing an in vivo RNA-interference screen. Eight ZAD-ZNF genes required for oogenesis were identified, and based on further characterization of the knockdown phenotypes, defects broadly consistent with functions in germ cell specification and/or survival, early differentiation, and egg chamber maturation were uncovered. These results provide a candidate pool for future studies aimed at functionalization of this large but poorly characterized gene family (Shapiro-Kulnane, 2021).
In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. This study shows that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. The RNA-degrading Super Killer complex and No-Go Decay factor Pelota were identified as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote (Blatt, 2021)
The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. This study used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization (Almeida-Oliveira, 2021).
Epithelia form protective permeability barriers that selectively allow the exchange of material while maintaining tissue integrity under extreme mechanical, chemical, and bacterial loads. This study in the Drosophila follicular epithelium reports a developmentally regulated and evolutionarily conserved process "patency", wherein a breach is created in the epithelium at tricellular contacts during mid-vitellogenesis. In Drosophila, patency exhibits a strict temporal range potentially delimited by the transcription factor Tramtrack69 and a spatial pattern influenced by the dorsal-anterior signals of the follicular epithelium. Crucial for growth and lipid uptake by the oocyte, patency is also exploited by endosymbionts such as Spiroplasma pulsonii. These findings reveal an evolutionarily conserved and developmentally regulated non-typical epithelial function in a classic model system (Row, 2021).
Sexual reproduction in internally fertilizing species requires complex coordination between female and male reproductive systems and among the diverse tissues of the female reproductive tract (FRT). This study reports a comprehensive, tissue-specific investigation of Drosophila melanogaster FRT gene expression before and after mating. Expression profiles were identified that distinguished each tissue, including major differences between tissues with glandular or primarily nonglandular epithelium. All tissues were enriched for distinct sets of genes possessing secretion signals that exhibited accelerated evolution, as might be expected for genes participating in molecular interactions between the sexes within the FRT extracellular environment. Despite robust transcriptional differences between tissues, postmating responses were dominated by coordinated transient changes indicative of an integrated systems-level functional response. This comprehensive characterization of gene expression throughout the FRT identifies putative female contributions to postcopulatory events critical to reproduction and potentially reproductive isolation, as well as the putative targets of sexual selection and conflict (McDonough-Goldstein, 2021b).
Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. This study demonstrates a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth (Stark, 2021).
In the germarium of the Drosophila ovary, developing germline cysts are surrounded by a population of somatic escort cells that are known to function as the niche cells for germline differentiation;(1) however, the underlying molecular mechanisms of this niche function remain poorly understood. Through single-cell gene expression profiling combined with genetic analyses, this study demonstrates that the escort cells can be spatially and functionally divided into two successive domains. The anterior escort cells (aECs) specifically produce ecdysone, which acts on the cystoblast to promote synchronous cell division, whereas the posterior escort cells (pECs) respond to ecdysone signaling and regulate soma-germline cell adhesion to promote the transition from 16-cell cyst-to-egg chamber formation. The patterning of the aEC and pEC domains is independent of the germline but is dependent on JAK/STAT signaling activity, which emanates from the posterior. Thus, a heterogeneous population of escort cells constitutes a stepwise niche environment to orchestrate cystoblast division and differentiation toward egg chamber formation (Shi, 2020).
In the germarium of the Drosophila ovary, 2 to 3 germline stem cells reside at the anterior tip, next to a cluster of cap cells. These cap cells secrete the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp) to promote stem cell self-renewal, thereby acting as the niche for germline stem cell maintenance. The immediate daughters of germline stem cells (GSCs), namely cystoblasts (CBs), move posteriorly as they differentiate: each CB divides 4 times with incomplete cytokinesis to form an interconnected 16-cell cyst in which one of the cells adopts an oocyte fate and the rest become supporting nurse cells (Shi, 2020).
During the differentiation process, these germline cysts are continuously surrounded by a group of somatic cells termed escort cells (ECs). These ECs send long protrusions to intimately interact with the germline cysts, but they do not move posteriorly along with the differentiating germline cysts. Instead, they appear to act as a flexible channel for the cysts to pass through (spanning from region I to IIa) until they reach the follicle stem cell (FSC) site (the boundary between the IIa and IIb regions), where each cyst starts to be surrounded by a single layer of follicle cells to form egg chambers. The FSC region contains 2-14 FSCs, as the exact number is still under debate. Recent work suggests that FSCs not only generate daughters that move posteriorly and differentiate into follicle cells but also give rise to daughters that move anteriorly and differentiate into ECs. The ablation of ECs or impairment of their protrusions causes blocked CB differentiation suggesting that ECs may provide an essential niche environment for germline cyst differentiation (Shi, 2020).
The EC population is likely heterogeneous. For instance, the anterior-most ECs that contact the cap cells specifically express Dpp and contribute to GSC self-renewal, although the ECs positioned next to the FSC site specifically secrete Wnt molecules to regulate FSC self-renewal, in addition to the distant source of Wnt signals from cap cells. Moreover, the ECs located near the IIa-IIb boundary might include some FSCs and early progenitors that are primed for EC differentiation. To better understand the heterogeneity of the EC population, fluorescence-activated cell sorting (FACS) was used to sort the ECs labeled by c587-GAL4, upstream activating sequence (UAS)-GFP (c587 > GFP, for short) from ovaries of 3- to 7-day-old females, and scRNA-seq was conducted using 10x Genomics technology. c587 > GFP marks all the ECs as well as FSCs and some early follicle progenitor cells. A total of 1,864 single cells were profiled after filtering out low-quality cells. Cells were sequenced to an average depth of 44,800 unique molecular identifiers (UMIs) and 2,470 genes per cell with less than 2% of reads for mitochondrial genes (Shi, 2020).
Data analysis using the R package Seurat3 partitioned the cells into 8 distinct clusters (using the top 30 principal components at a resolution of 0.4). These clusters were visualized with a uniform manifold approximation and projection (UMAP) plot. A number of known cell makers were used to characterize these different cell clusters. Nearly all of the cells were negative for the germline-specific marker vasa, suggesting no significant contamination from germline cells. However, there appeared to be some contamination of cap cells, represented in the relatively small cluster 6, which were positive for the cap cell marker bab1. Two known follicle cell markers Fasciclin 3 (Fas3) and cut (ct) enabled definition of clusters 2-5 as follicle cell clusters, which were referred hereafter as a combined 'follicle cell cluster'. The remaining two large clusters 0 and 1 were likely ECs that consisted of totally 1,166 cells. As each germarium contains approximately 40-50 escort cells, the total number of profiled single ECs is more than 23 times the number of total ECs per germarium. The differentially expressed genes found in clusters 0 and 1 were subsequently assessed with Gene Ontology (GO) enrichment analysis along with that from the follicle cell cluster using Metascape (Shi, 2020).
Of note, the above analysis failed to identify a specific cluster for FSCs. This study also failed to identify any cell clusters that display increased or decreased mitotic activity using the cell-cycle scoring and regression analysis, a method that has been used to identify stem cell populations. This indicates that the mitotic activity between FSCs and ECs is not sufficiently different to be separated by this analysis. It is speculated that FSCs might be mixed within the cluster 0 cells, a possibility that will be discussed shortly (Shi, 2020).
GO analysis suggested several overlapping as well as distinct biochemical and developmental processes at work by these three different groups. As expected, the FC cluster showed specific activity in chorion-containing eggshell formation (such as Cp15, Cp19, and Fcp3C in clusters 2, 4, and 7, in particular for 4 and 7) and other processes that are known to be involved in later stages of oogenesis. Notably, the cluster 1 showed putative activity for the ecdysone biosynthetic process, although the cluster 0 showed enrichment of genes involved in vitelline membrane formation (such as Vm32e, Vm26Aa, and Vml). To further evaluate the functional relevance in germline cyst differentiation, these differentially expressed genes found in these three clusters were subjected to a small-scale RNAi screen for their requirement in ECs for egg production, and these with failed egg production were analyzed further for defects in early germline development. Genes whose depletion showed defects in the germarium were considered with high confidence of functional relevance, and the gene expression changes for these genes were summarized and presented as a heatmap. Comparative gene expression analysis among those selected genes further supports functional continuation among the three cell clusters: cluster 0 appeared to be at an intermediate state between cluster 1 and the follicle cell cluster, as about half of the selected candidate marker genes overlapped with cluster 1 and the other half overlapped with the follicle cell cluster. Of note, genes involved in ecdysone biosynthesis, including phantom (phm) and shadow (sad), were shown as cluster-1-specific genes (Shi, 2020).
To map out the spatial distribution of clusters 0 and 1, fluorescence in situ RNA hybridization (FISH) was used to determine the expression pattern of the cluster-1-specific phm and IA-2, which is expressed in cluster 0 and in a subset of follicle cells. Consistent with the scRNA-seq results, the FISH signal for phm was specifically detected in ECs at region I, but not region IIa, of the germarium, suggesting that cluster 1 represents ECs located at the anterior region roughly corresponding to region I. Also consistent with the single cell results, the FISH signal for IA-2 was specifically detected in ECs in the region IIa as well as FSCs and some early follicle progenitor cells. Taken together, these results suggest that cluster 1 and 0 represent two spatially distinct EC populations located, respectively, roughly at region I and IIa of the germarium. As mentioned earlier, the cluster 0 cells, namely ECs at region IIa, possibly contain some FSCs and their immediate progenies, as the IIa-IIb region is considered be circumvented by three lines of FSCs. The lineage-tracing experiment conducted later in this study also supports the idea that ECs at region IIa can be derived from FSCs. But for simplicity, the cluster 1 cells are termed anterior ECs (aECs) and the cluster 0 cells as posterior ECs (pECs) (Shi, 2020).
The scRNA-seq analysis therefore allowed identification of two major subpopulations of ECs that are separately distributed in the germarium, and the distinct as well as continuous gene expression characteristics among aECs, pECs (which may include FSCs), and early follicle cells indicates functional separation as well as continuation among these 3 groups of cells (Shi, 2020).
After screening a collection of GMR- and NP-GAL4 transgenic/enhancer trap strains, GMR25A11-GAL4 (aEC-GAL4 for short), which carries an enhancer element for the Wnt4 gene, was identified as a possible specific GAL4 driver line for aECs. Wnt4 has also been reported to be expressed in ECs or subsets of ECs. Additionally, wun2NP3026-GAL4 (pEC-GAL4 for short) driver, which appeared to drive UAS-GFP expression specifically in pECs and cap cells, was identified, although weak GFP expression was also found in aECs in some germaria when the image was overexposed. The EC-specific expression of these two GAL4 lines was also confirmed by co-staining with PZ1444-lacZ, a known EC marker. Further examination of the patterns of these two GAL4 lines in relationship with the stages of the germline cysts revealed that aEC-GAL4+ cells were associated with 4-cell-or-less cysts, although pEC-GAL4+ cells were associated with 8- and 16-cell cysts. qRT-PCR analysis of the sorted aEC-GAL4; UAS-GFP (aEC > GFP+) and pEC-GAL4;UAS-GFP (pEC > GFP+) cells confirmed that aEC > GFP+ cells have relatively higher expression of phm and pEC > GFP+ cells have relatively higher expression of IA-2. Note that the qRT-PCR results also showed that Wnt4 expression was higher in aECs than pECs, which is consistent with the expression pattern of its GAL4 reporter used in this study. Collectively, these results suggest that these two GAL4 lines largely mark the scRNA-seq-classified aEC and pEC populations, respectively (Shi, 2020).
Cell ablation experiments were conducted with the aEC-GAL4 and pEC-GAL4 lines to determine the specific requirements of these subsets of ECs for germline differentiation. Conditional expression of an apoptosis inducer reaper (rpr) for 7 days either in aECs or pECs effectively ablated these cells. As a positive control, total EC ablation using the c587-Gal4 driver caused a complete blockage of CB differentiation, leading to a strong CB accumulation phenotype. Ablation of aECs caused mild accumulation of CB with reduced populations of 2-, 4-, and 8-cell cysts in the germarium, suggesting that aECs function in the progression of CB differentiation into 8-cell cysts. This phenotype is considerably milder than that caused by total EC ablation, suggesting that the remaining pECs might be able to somehow partially compensate the loss of aECs. The ablation of pECs caused specific and strong accumulation of 8-cell cysts in the germarium. In addition, pEC ablation resulted in 16-cell cysts at region IIb, which were unable to be properly encapsulated by follicle cells to form egg chambers, leading to a swollen germaria (increased circumferential length at region IIb) phenotype. Collectively, these results indicate that aECs functionally impact cyst cell division during CB differentiation into 8-cell cysts, whereas pECs continue to support cyst division through to the final 16-cell cyst formation. In addition, pECs may help to facilitate the developmental transition between pEC-surrounded 16-cell cyst and follicle cell-encapsulated egg chambers (Shi, 2020).
It was also found that pECs in particular may impact the survival of the developing germline cysts: the frequency of apoptotic cysts, especially those at the posterior region, was significantly increased following pEC ablation. Given that one oocyte is specified in each newly formed 16-cell cyst, whether ablation of pECs could affect oocyte specification was examined. The oocyte marker Orb normally starts its expression in all cells in 8-cell cysts and soon confined to two pre-oocytes and then finally one oocyte in 16-cell cysts. Interestingly, in both aEC- and pEC-ablated germaria, similar to normal patterns, Orb was weakly expressed in all of the 8-cell cysts examined; one Orb+ oocyte or two Orb+ pre-oocytes were invariably present in the 16-cell cysts examined. Thus, it appears that pECs are not essential for oocyte differentiation and specification, as long as the germline cyst has developed to the 16-cell stage (Shi, 2020).
The expression of two ecdysone biosynthetic genes phm and sad in aECs indicates the ability of aECs to produce ecdysone. Ecdysone signaling has been previously implicated in regulating adult Drosophila oogenesis. In the ovary, although ecdysone is found to be mainly produced in stage 8 and later egg chambers, it is speculated that aECs may provide a local source of ecdysone to regulate early stages of germline development. phm or sad were conditionally depleted in either aECs or pECs by RNAi using the aEC- and pEC-specific GAL4 drivers, and the effect on the germline development after 7 days of treatment was examined. Conditional depletion of phm in aECs, but not pECs, caused significant increase of 8-cell cyst per germarium and concurrent decline of earlier stages of germline cysts. Similar results were found for sad RNAi. This phenotype is similar to that caused by aEC ablation in aspects of significantly shrunk I and IIa regions of the germarium; however, aEC ablation shows increased CBs and decreased 8-cell cysts, opposite to that caused by phm depletion. The difference in CBs is possibly because of different effect on GSC maintenance and proliferation in these two different conditions, as conditional depletion of phm or sad in aECs also slightly reduced the GSC number in each germarium. The increase of 8-cell cysts in aEC > phm/sad-RNAi germaria is probably due to a non-cell-autonomous effect on pECs, which will be described shortly. It is concluded that cyst cell division promoted by aECs is mediated by ecdysone signaling: aECs produce ecdysone, which then acts on the associated germline cells to promote synchronous cell division toward 8-cell cysts (Shi, 2020).
Interestingly, the single-cell data also revealed that ftz-f1 and Eip78c, two ecdysone target genes, were both expressed weakly in pECs and strongly in follicle cells; neither gene was expressed in aECs. Given the finding that the Ecdysone receptor (EcR) was expressed in all ECs, pECs can potentially receive ecdysone signals produced from aECs and other sources. Eip78c-GFP, a genomic transgenic reporter fused with GFP, was used to further examine Eip78c expression in the germarium. For the germline cells in regions I and IIa, GFP was specifically expressed in GSCs, CBs, and 16- cell cysts. For somatic cells, GFP was detectable only in cap cells, pECs, and follicle cells. An EcR activity reporter line (UAS-Stinger; hs-GAL4-usp.LBD) was used to detect EcR activity in the germarium, and this reporter line showed a largely similar expression pattern to Eip78c-GFP. Therefore, in the anterior regions of the germarium, relatively high levels of EcR activation are found in GSCs and CBs in the germline and found in pECs and follicle cells in the soma. These results are consistent with the idea that local production of ecdysone by aECs activates EcR in GSC and CB to promote cell division, and the observations also suggest that this local source of ecdysone also regulates EcR activity in pECs (Shi, 2020).
To investigate whether the observed EcR activity in pECs impacts the function of pECs, EcR was depleted specifically in pECs via RNAi and such depletion caused a specific accumulation of 8-cell cysts in EC-surrounding regions. In contrast, depletion of EcR in aECs did not cause any visible phenotypes. Depletion of ftz-f1 in pECs caused a similar 8-cell cyst accumulation phenotype, indicating that ftz-f1 is an important downstream mediator of EcR signaling in pECs; again, depletion of ftz-f1 in aECs did not yield any obvious phenotypes. EcR or ftz-f1 depletion in pECs also caused a swollen germarium phenotype, as some 16-cell cysts appeared to be stagnant at region IIb and were not timely surrounded by the follicle cells to form egg chambers. It therefore appears that the EcR activity in pECs can mediate two separate roles of pECs-promoting 16-cell cyst formation from 8-cell cysts and promoting the developmental transition between 16-cell cysts and egg chamber formation (Shi, 2020).
As some FSCs are possibly mixed into the pEC-GAL4+ cell population, genetic analysis performed with the pEC-GAL4 driver could potentially reflect the function by FSCs rather than pECs. Cell lineage tracing experiments conducted with pEC-GAL4 indeed revealed that pEC-GAL4+ cells contained FSC activity. This contamination is considered minimal, as the majority of this population of cells represents the cells escorting 8- and 16-cell cysts, and these cells have low mitotic activity. Nevertheless, to evaluate the functional contributions of FSCs in the observed phenotypes, the c306-GAL4 driver, which is known to be expressed in FSCs and early FCs, was used for genetic analysis. It was found that ftz-f1 depletion driven by the c306-GAL4 driver did not cause any obvious 8-cell cyst accumulation at the IIa/IIb boundary or any obvious defects in 16-cell cyst-to-egg chamber transition, and EcR depletion only caused a very mild accumulation of 8-cell cysts, in contrast to much stronger phenotypes caused by EcR or ftz-f1 depletion driven by the pEC-GAL4 driver. Therefore, the observed phenotypes associated with the pEC-GAL4 driver can be largely attributed to the functional perturbation of pECs, but not FSCs (Shi, 2020).
During the transition leading to egg chamber formation, the 16-cell cysts are immediately surrounded by a layer of follicle cells as they are losing contact with pECs. Although the exact process of this transition is still unclear, it is reasonable to speculate that cell-cell adhesion might influence this developmental transition. Germline-soma interaction via DE-cadherin (DE-cad)-mediated cell adhesion is known to regulate germline development, as for example in stem cell maintenance and oocyte positioning (Shi, 2020).
To determine whether EcR activity in ECs regulates cell adhesion, DE-cad expression was examined in EcR-depleted pECs. Within the I and IIa regions of wild-type germaria, the strongest DE-cad expression was observed in cap cell clusters, with prominent expression additionally evident in the ring canals of 8- and 16-cell cysts, similar to results reported previously. DE-cad expression was also found in pECs, but not aECs, at the interface between ECs and germline cysts, including the soma-germline interface at the IIa/IIb boundary region. Strikingly, depletion of EcR in pECs caused a dramatic downregulation of DE-cad expression, generally at the interface between soma and 16-cell cysts, including the soma-germline interface at the IIa/IIb boundary region. In contrast, depletion of EcR in aECs did not cause any obvious changes in DE-cad expression in the germarium. Depletion of ftz-f1 in pECs caused a similar downregulation of DE-cad at the soma-germline interface; moreover, ftz-f1 depletion in aECs did not cause any significant changes in DE-cad expression in the germarium (Shi, 2020).
It is worth noting that, although the downregulation of DE-cad at soma-germline interface was obvious, there was no significant downregulation in DE-cad expression in the ring canals of 8-cell and 16-cell cysts in the above EcR RNAi or ftz-f1 RNAi germaria, showing that the depletion of EcR or ftz-f1 in pECs specifically impacts DE-cad-mediated cell adhesion between pECs and germline cysts. These observations collectively indicate that the EcR activity that was observed in pECs regulates DE-cad-mediated soma-germline cell adhesion and additionally it is suggested that this interaction impacts the formation of follicle-cell-encapsulated cysts as they develop from EC-surrounded 16-cell cysts (Shi, 2020).
As DE-cad-mediated cell adhesion has been implicated in controlling directional cell migration, such as border cell-nurse cell adhesion during border cell migration, it is tempting to speculate that adhesive interaction between the pEC and the 16-cell cyst may help to guide the 'migration' of the cyst toward posterior, thereby facilitating the transition between 16-cell cysts and egg chamber formation. To further test this speculation, the adhesive interaction was disrupted by depleting DE-cad gene (shotgun, shg) via RNAi, by depleting α-catenin via RNAi, or by expressing a truncated form of Shg that is unable to bind β-catenin (ShgΔβ) and thus has a dominant negative effect on cell adhesion in pECs. All of these genetic manipulations caused similar cyst transition defects, as many 16-cell cysts at regions IIb and III were not timely encapsulated by follicle cells and appeared stagnant at region III of the germarium. This stagnant phenotype associated with 16-cell cysts, characterized additionally by the tilted shape and compaction, were similarly observed in germaria depleted with EcR or ftz-f1 in pECs. Interestingly, there was no apparent increase in egg chamber fusion as these 16-cell cysts developed, but egg chambers with incomplete follicle cell encapsulation could be observed occasionally. Thus, DE-cadherin-mediated cell adhesion between pECs and germline cysts is functionally important for the developmental transition between pEC-surrounded 16-cell cyst and follicle-cell-encapsulated egg chambers. Of note, unlike ecdysone signaling, the adhesive interaction appears to be not required for the final synchronous division for 16-cell cyst formation from 8-cell cyst (Shi, 2020).
Next the mechanism underlying aEC/pEC patterning was explored. As aECs and pECs are associated with germline cells at different developmental stages, it was speculated that the developmental stage of a given germline cyst may determine the spatial identity of its physically associated ECs. This using two approaches. In the first approach, GSC differentiation was induced by overexpressing a differentiation-promoting gene, bam, so that the stem cells and early germline cysts that located at the anterior region were forced to differentiate into late-stage germline cysts in situ, before eventually moving out of the germarium. During this process, the aEC/pEC cell identity remained intact. When all the germ cells were gone, the remaining ECs were physically clustered together, but their original anterior/posterior identity nevertheless remained. In the second experimental approach, aEC and pEC markers were examined in bgcn mutant germaria, wherein all of the germline cells are arrested at a CB-like stage. Interestingly, although excessive accumulation of proliferative CB-like cells caused swollen and deformed germaria, the relative positioning of aECs and pECs still remained. Therefore, the patterning of aEC and pEC domains is not dependent on the stage information of the physically associated germline cysts. In fact, the maintenance of aEC/pEC domains, at least in a short run, does not even require the existence of germline cells (Shi, 2020).
Lacking a germline influence, it follows that the patterning of aEC and pEC domains is apparently regulated via a somatic mechanism. Previous studies have revealed multiple signal molecules that are secreted from the cells either anterior or posterior to the EC resident regions, such as BMP, Hedgehog (Hh), and Wnt produced from the cap cells (anterior) and Upd produced from early follicle cells (posterior). These signals generated from one pole will defuse toward another end, and the resultant signaling activation gradient may underlie the patterning of the aEC/pEC domains. However, BMP, Hh, and Wnt can also be produced in ECs, and the expression of various previously described signaling activation reporters does not support an obvious gradient of Hh or Wnt activity within the EC populations, except in the regions immediately next to the FSC site. Interestingly, consistent with a previous observation, it was found that Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway activity, revealed by a 10XSTAT-GFP reporter, displayed a gradient pattern emanating from early follicle cells to pECs. This observation suggests that JAK/STAT signaling might participate in the aEC/pEC patterning or somehow otherwise affect pEC fate (Shi, 2020).
Pursuing this, the JAK/STAT signaling activity was genetically manipulated in either aECs or pEC by expressing the JAK kinase Hop to activate the pathway, and expressing a dominant-negative form of the receptor Dome to inhibit the pathway. Neither activation nor inhibition of the JAK/STAT pathway affected the identity, distribution, or the total number of aECs. However, inhibition of JAK/STAT signaling in pECs caused a significant reduction of pEC > GFP+ cells. In contrast, JAK/STAT signaling activation in pECs did not alter any pEC > GFP+ cell phenotypes. Considering the findings suggesting that JAK/STAT activity may potentially affect EC proliferation or survival, the total EC number was calculated in each germarium using PZ1444-lacZ, and quantitative analysis indicated that the JAK/STAT pathway does not apparently have a significant impact on the total number of ECs in the germarium, rather, JAK/STAT signaling may be required for pEC fate maintenance (Shi, 2020).
Considering that JAK/STAT and EcR activities both occur in pECs, it was asked whether there is any regulatory or functional connection between JAK/STAT and EcR in pECs. Depletion of EcR in pECs does not seem to affect JAK/STAT activity in pECs, as the 10XSTAT-GFP reporter expression remained unaltered; however, inhibiting JAK/STAT by expressing DomeDN in pECs caused significant downregulation of Eip78c-GFP expression in pECs, suggesting that JAK/STAT is required for EcR activity in pECs. Not surprisingly, inhibition of the JAK/STAT pathway also caused a dramatic decline of DE-cad expression at the interface between pECs and germline cysts. Forced activation of JAK/STAT activity by overexpressing hop in pECs did not significantly affect DE-cad expression, and this forced JAK/STAT activation also failed to restore the downregulated DE-cad level caused by EcR depletion. These results suggest that JAK/STAT functions through ecdysone signaling to regulate cell adhesion. Collectively, these results led to the proposal of a model as following: the JAK/STAT activity, which is active in pECs, allows pECs to be responsive to ecdysone signaling, whose activity then regulates cell adhesive interaction between germline and soma to promote the transition between 16-cell cyst and egg chamber formation (Shi, 2020).
Collectively, this study supports that ECs, FSCs, and early follicle cells, guided by cell-to-cell signaling communications, furnish a progressive microenvironment for the stepwise differentiation of CBs toward egg chamber formation. The aECs produce local ecdysone that acts on the germline to promote synchronous cyst division; the pECs continuously support a final cyst division. Further, owing to their JAK/STAT activity provided by early follicle cells, the pECs are responsive to ecdysone and thereby undergo increased DE-cad-mediated soma-germline interactions, which facilitate the developmental transition toward egg chamber formation. Consistent with a progressive nature for the microenvironment, the aEC, pEC (which likely includes FSCs), and follicle cell clusters depicted in the UMAP plot are continuous and overlapping. The lineage relationships among these groups of somatic cells was followed by conducting cell lineage tracing experiments using the newly characterized aEC-GAL4 and pEC-GAL4, as well as two previously reported FSC and follicle cell driver lines c306-GAL4 and 109-30-GAL4. The aEC-GAL4+ cell-derived progenies were able to slowly expand to the pEC resident region over a 2-week period; conversely, the pEC-GAL4+ cell-derived progenies were also able to expand to the aEC-resident region but happened in a much slower manner; the c306-GAL4+ and 109-30-GAL4+ cell (presumptively FSCs in both cases)-derived progenies were able to slowly migrate to pEC-resident region and occasionally aEC-resident region over 2 weeks. These observations, along with numerous published observations, collectively lead to an inclusive model for the homeostatic regulation of aECs and pECs: the homeostasis of aECs and pECs is maintained through multiple mechanisms, including self-duplication, fate exchange, and FSC activity. In addition to the recent observations that ECs and follicle cells may share a common developmental origin (Slaidina, 2020; Reilein, 2020), the intimate cell lineage relationships and fate interchangeability also support the idea of functional continuity among aECs, pECs, FSCs, and follicle cells that collectively provide a progressive niche environment for orchestrating germline differentiation (Shi, 2020).
The successful identification of two subgroups of ECs in this work is likely due to the large quantity of ECs used in the single-cell analysis. This probably explains why this is missed in some similar work done by others. However, it bears emphasis that, although the present study empirically supports the classification of ECs into two major subpopulations, each of these EC subpopulations could potentially be further divided into additional subtypes. Such further classification may be limited by the 10x Genomics single-cell approach, through which potentially informative genes expressed at low levels could be missed. For instance, Wnt4, which is known to be expressed in ECs, or dpp, which is expressed in the anterior-most ECs, was not detected in the single-cell data (Shi, 2020).
The finding of a progressive niche for germline development in Drosophila is likely conserved in mammals: a recent study revealed the presence of escort-like somatic cells and the derived granulosa cells in mouse fetal ovaries and showed that these cells interact with germ cells and developing germline cysts. Further, in the mammalian testis, the Sertoli cells, which support the entire process from spermatogonia differentiation to sperm formation, have long been proposed to have functional subdomains. It is proposed that a progressive niche environment may be a common scheme for guiding the development and differentiation of germline cells and possibly other types of adult stem cells (Shi, 2020).
Small cell clusters exhibit numerous phenomena typically associated with complex systems, such as division of labour and programmed cell death. A conserved class of such clusters occurs during oogenesis in the form of germline cysts that give rise to oocytes. Germline cysts form through cell divisions with incomplete cytokinesis, leaving cells intimately connected through intercellular bridges that facilitate cyst generation, cell fate determination and collective growth dynamics. Using the well-characterized Drosophila melanogaster female germline cyst as a foundation, this study presents mathematical models rooted in the dynamics of cell cycle proteins and their interactions to explain the generation of germline cell lineage trees (CLTs) and highlight the diversity of observed CLT sizes and topologies across species. Competing models of symmetry breaking in CLTs were analyzed to rationalize the observed dynamics and robustness of oocyte fate specification, and highlight remaining gaps in knowledge. How CLT topology affects cell cycle dynamics and synchronization were analyzed and mechanisms of intercellular coupling are highlighted that underlie the observed collective growth patterns during oogenesis. Throughout, similarities across organisms are described that warrant further investigation and comments are made on the extent to which experimental and theoretical findings made in model systems extend to other species (Diegmiller, 2022).
Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B/H2A/H2Av accumulate on lipid droplets (LDs), cytoplasmic fat storage organelles. Without LD-binding, maternally provided H2B/H2A/H2Av are absent, but how LDs ensure histone storage is unclear. Using quantitative imaging, this study uncover when during oogenesis these histones accumulate, and which step of accumulation is LD-dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone-anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. These findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites (Stephenson, 2021).
Early animal embryogenesis often exhibits rapid cell cycles dominated by DNA replication, mitosis and little to no transcription. Drosophila is a dramatic case where the first 13 nuclear divisions occur every 8-20 min. This speed poses a challenge for histone biology: demand increases exponentially, yet major regulatory mechanisms that control histone expression are unavailable. To meet this demand, many embryos inherit maternally synthesized histones. This study examined the origin of H2Av, H2B and H2A stockpiles in Drosophila (Stephenson, 2021).
Expression of histone messages is increased during S10, but it was unclear when the complementary maternally deposited histone proteins accumulate. A methodological challenge is that enterocytes (ECs) have polyploid nuclei. Each of the nurse cell (NC) nuclei are estimated to contain about 500 times more DNA than diploid nuclei; the 900 follicle cells contain DNA levels about eight times higher than a diploid nucleus. Thus, the histones needed to package NC and follicle cell chromatin are at least tenfold more abundant than the histone stockpile of newly laid embryos, which are estimated to be the equivalent of 1000 diploid nuclei. Detecting accumulation of the oocyte histone stockpile in addition to other histones in the EC is therefore challenging (Stephenson, 2021).
This study followed H2Av accumulation using an imaging approach that specifically quantifies histone signal in the NC and oocyte cytoplasm. H2Av already accumulates in the cytoplasm of S9 NCs and is associated with lipid droplets (LDs). An intriguing possibility is that this LD-associated H2Av pool in the early-stage NCs might support NC endoreplication. This H2Av-LD association likely brings some H2Av into the oocytes, but quantitation indicates that transfer from NCs contributes at most one fifth of the final H2Av pool in mature oocytes. These data are consistent with the possibility that the majority of the ooplasmic H2Av is synthesized in the oocyte; reduced H2Av levels in Jabba−/− S12 oocytes may represent early loss by degradation rather than defective transfer from NCs (Stephenson, 2021).
After dumping, total H2Av levels continue to rise from S12 through S14. It is proposed that two mechanisms contribute to this rise. First, the translational efficiency of H2Av mRNA is upregulated from S12 to S14. Second, Jabba increases from S12 to S14. As more Jabba protein becomes available, it can presumably recruit more H2Av to LDs and protect it from degradation (Stephenson, 2021).
H2A and H2B levels in embryos are Jabba dependent (Li, 2012), and the pattern of H2B accumulation during oogenesis resembles that of H2Av. Currently, good tools to determine H2A accumulation are lacking, but it is predicted to will follow the same pattern, as canonical histones are typically similarly regulated. It is proposed that increasing H2Av, H2B and H2A accumulation during late oogenesis establishes an LD-bound histone depot for the early embryo (Stephenson, 2021).
It will be interesting to determine whether H3 and H4 accumulation follows a similar pattern. As canonical histones, their transcriptional and translational regulation is likely similar to that of H2A and H2B. However, they are not LD associated nor are their embryonic levels Jabba dependent, so there must be mechanistic differences (Stephenson, 2021).
Newly laid Jabba−/− eggs have lower H2A, H2B and H2Av levels than wild type. This divergence is established during late oogenesis; H2Av and H2B levels rise in the wild type but drop in Jabba−/−. Experiments to inhibit proteasome activity reveal that histone degradation plays a major role in bringing about this difference. As high MG132 (proteasome inhibitor) concentrations arrest development, it is infered that under conditions that allow EC development to S14, degradation is only partially inhibited. Yet even with partial inhibition, almost half of the H2Av normally lost in Jabba−/− is retained. It is concluded that Jabba prevents histone turnover and that a major contributor to turnover is a proteasome-dependent pathway. The remaining turnover not prevented by drug treatment may be due to a proteasome-independent mechanism (Stephenson, 2021).
In the wild type, proteasome inhibition did not increase the H2Av pool beyond levels in untreated ECs. This observation was surprising as 4x Jabba females can accumulate more H2Av than wild type, which presumably indicates that even the wild type produces excess H2Av that is degraded if not protected by Jabba. It was reasoned that the direct effects on proteasome turnover are balanced out by indirect effects that compromise histone or Jabba synthesis. Indeed, high concentrations of the inhibitor abolish any rise in H2Av.
Turnover of excess histones by the proteasome is well established in yeast. It is one of numerous mechanisms that prevent accumulation of free histones and the resulting cytotoxicity. It is proposed that H2Av turnover in late oogenesis is due to this general protective machinery; Jabba allows oocytes to deploy this safety feature while also accumulating H2Av needed to provision the embryo. Accordingly, Jabba determines the H2Av pool that is protected and any excess is recognized as a potential hazard. Because the mechanism that targets excess H2Av for proteasomal degradation is not known, it cannot yet be tested what type of damage this mechanism guards against (Stephenson, 2021).
The data suggest that Jabba prevents degradation by physically protecting H2Av. First, H2Av levels scale with Jabba dose, and H2Av levels beyond normal can be achieved by simply doubling Jabba levels. Second, a version of Jabba that is mislocalized to the NC nuclei and not present in oocytes is unable to support high H2Av levels. Finally, a Jabba mutant unable to bind to H2Av resulted in H2Av levels indistinguishable from those in Jabba−/− (Stephenson, 2021).
To further unravel how Jabba prevents degradation, it will be necessary to identify the machinery that targets excess H2Av to the proteasome. Previous work has identified E3 ligases promoting H3 and H4 turnover, but those for other histones remain uncharacterized. Jabba may physically protect H2Av by shielding a ubiquitylation site. This remains to be tested. An alternate hypothesis is that histone degradation in oocytes occurs independently of ubiquitylation. Intriguingly, during development of the mammalian male germline, histone turnover occurs via ubiquitin-independent proteasome-dependent degradation (Stephenson, 2021).
In embryos, H2Av exchanges between LDs. If similar exchange occurs in oocytes, it is unclear how transient interactions with Jabba are sufficient to prevent H2Av degradation. It is speculated that either the transit time is negligible relative to the time H2Av spends interacting with Jabba or that cytosolic H2Av is accompanied by a chaperone.
LD binding promotes availability of Jabba and, consequently, of histones
Our analysis provides a first answer to why some histones are stored on LDs, while others are apparently stored in the cytoplasm. Jabba, the protein necessary to stabilize the H2A, H2B and H2Av ooplasmic pool, becomes trapped in NC nuclei if it is not LD bound. Thus, it is absent from the oocyte and cannot perform its protective function. It is proposed that LD binding ensures proper intercellular transport of Jabba, safeguarding its ability to function in the ooplasm (Stephenson, 2021).
H3 and H4 ooplasmic stores are presumably bound to a partner that prevents their degradation, perhaps the histone chaperone NASP. As these histones are apparently cytoplasmic, it is unclear how they and their binding partners avoid mislocalization to NC nuclei (Stephenson, 2021).
It is also unclear why histones are stored on LDs rather than another cytoplasmic structure. A priori, any cytoplasmic organelle that is transferred to oocytes could suppress the NC nuclear import of the histone anchor and promote transport into the oocyte. The large surface area of LDs may provide a readily available and, at these developmental stages, metabolically inert platform for recruitment. Other organelles may not have enough storage capacity or might be functionally impaired by histones on their surface. Alternatively, LD association may be an evolutionary accident and many other organelles might in principle be able to store histones. Identification of a Jabba region sufficient for histone binding will make it possible to address this question in the future (Stephenson, 2021).
But why is JabbaHBR mislocalized to NC nuclei? It is suspected that the histone-binding ability of JabbaHBR leads to its mislocalization, dragged along by histone nuclear import or retained in nuclei via chromatin binding. As histone binding is important to protect against degradation, mislocalization cannot be avoided unless JabbaHBR is anchored outside the nucleus. This idea could not be tested, as JabbaHBR that is unable to interact with histones still accumulates in nuclei, in cultured cells and in NCs. It is hypothesized that a cryptic nuclear localization signal in JabbaHBR promotes nuclear transport even without histone binding (Stephenson, 2021).
This analysis may shed light on how LDs regulate other proteins. LDs can transiently accumulate proteins from other cellular compartments. This has been particularly documented for proteins involved in nuclei acid binding and/or transcriptional regulation: MLX and Perilipin 5 can either be present on LDs or move into the nucleus to regulate transcription. The bacterial transcriptional regulator MLDSR is sequestered to LDs under stress conditions. As for Jabba and H2Av, LD association of such 'refugee proteins' may prevent their premature turnover or may promote their delivery to the correct intra- or intercellular location. A role for LDs in protein delivery to distant cellular compartments might be important in neurons where recent discoveries suggest important, but largely uncharacterized, roles for LDs (Stephenson, 2021).
Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. It was recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. This study tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, it was found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. It was hypothesized that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity (Neiswender, 2021).
KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).
This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).
Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).
In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok1 and enok2 ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).
Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300 . However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).
The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).
In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).
Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. This study demonstrates that Hecw (CG42797), a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons (Fajner, 2021).
