The Uncoordinated (Unc) gene product, a potential ortholog of orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. This study shows that evenly spaced Unc-GFP spots are present in the eye imaginal discs of third instar larvae. These spots are restricted to the R8 photoreceptor cell of each ommatidium in association with mother centrioles. This finding is unexpected since the Drosophila eye is of rhabdomeric type and should lack ciliary structures (Gottardo, 2016).

From its initial specification in the morphogenetic furrow, to its adult service as a photosensor, a developing Drosophila R cell plays many roles, calling upon a sequence of conserved, core cell functions that target the developmental task at hand. In the last quarter of pupal life that task is the enormous expansion of the photosensory membrane via secretory delivery of membrane rich in rhodopsin and allied elements of phototransduction. This study shows a corresponding expansion of stacked rER meets this secretory challenge; at this developmental stage, R cells are typical of cells devoted to intensive secretion, 'professional' secretory cells, generally. As seen with other professional secretory cells, this study found that R cells require Ire1 for normal ER expansion and differentiation. When normal cells proliferate rER stacks, Ire1 mutant ER proliferates in a dense, chaotic tangle of reticulon-rich tubules. The mechanisms by which Ire1 supports normal R cell ER differentiation remain unknown and in view of its multiple and far-reaching effectors are likely to be multiple. Shown in this study recapitulated in R cells, a common theme in Ire1 regulation of ER differentiation is a requirement in anticipation of secretory activity, suggesting Ire1 builds in secretory capacity as part of normal organelle programming. It is unlikely Ire1 acts alone and temporal overlap of Ire1 activity, shown in this study, and also by (Coelho, 2013), with that of PERK, a second conserved UPR pathway suggests they may cooperate in ER programming. The profuse tangle of reticulon-rich tubules that arises during peak secretory effort, resembles the pathology seen in Ire1 mutant yeast upon ER stress: in response to unfolded protein stress, normal yeast proliferate ER sheets but, although normal in the absence of stress, stressed Ire1 mutant yeast expand ER in dense, reticulon-rich tangles. Together, results suggest the possibility that Ire1 contributes essential shaping activity to a program of ER expansion and, in its absence, an unbalanced drive expands dysmorphic ER (Xu, 2016).

Shown in this study, and previously (Coelho, 2013), Ire1 loss compromises Rh1 production and secretory delivery needed to build the rhabdomere. Prior work has shown that Ire1's contribution to these tasks is Xbp1-independent, which is in accord with the current study, and now has been extended to ER shaping; R cell ER is normal in severe Xbp1 hypomorphs. Previously, it has been shown that Ire1 contributes to Rh1 delivery via degradation of fatty acid transporter, fatp, mRNA: Ire1 loss elevates fatp mRNA and its reduction using RNAi rescues Rh1 delivery (Coelho, 2013). As elevated levels of phosphatidic acid (PA) have been shown to disrupt R cell apical membrane transport, it is proposed that increased fatp elevates PA and thus degrades Rh1 delivery (Coelho, 2013). Abnormal, expanded rER is seen in R cells with increased PA, but appears distinct from the tangled tubular ER seen in this study. Tubulated ER seen in this study also differs from the dilated ER lumens commonly noted when misfolded secretory protein products over-accumulate, e.g., in Akita mice that accumulate misfolded proinsulin, and distinct from the strongly amplified, well-formed rER seen in ninaA mutants caused by lumenal Rh1 accumulation. Failure to assemble expanded rER sheets with accumulation of irregular, ribosome-poor ER tubules in Ire1 mutant R cells is reasonably the ultrastructural substrate for the severe reduction of Rh1 levels and growth deficit generally; without a definitive measure of reduced Rh1 synthesis, it is possible that reduced Rh1 levels are attributable to enhanced ER-Associated Degradation (ERAD) in an out of control ER. Loss of a normal SRC in Ire1 mutants plausibly contributes to the failure to deliver secretory traffic to the growing rhabdomere. Collapse of normal ER morphology is thus catastrophic for multiple cell activities (Xu, 2016).

The mechanism by which lower temperature rescues ER differentiation in Ire1 mutant cells remains to be determined, but may be connected to numerous observations showing basal, Ire1-independent, ER capacity supports many aspects of normal cell development and physiology. Indeed, despite profound ER disruption during peak rhabdomere growth, mutant R cells are viable and execute a wide range of normal cell physiologies, including normal cell polarity, cell fate specification and the complex choreography that assembles ommatidia. Similarly, Ire1 null nematodes are viable and morphologically normal, but die when challenged with mutations in other UPR branches or tunicamycin. Mouse hepatocytes lacking Ire1 α show reduced rER content but are otherwise phenotypically normal; however upon ER stress they fail to maintain lipid homeostasis. The gut of mice lacking Ire1beta is phenotypically normal, but sensitized to experimental colitis. In the absence of stress, ER morphology is normal in yeast ire1 mutants. It is speculated that when pupal development is slowed more than, twofold, 9-10 days at 19°C, versus 4 days at 25°C, basal ER capacity meets secretory demand (Xu, 2016).

The distribution of peripheral ER between sheet and tubule domains has been likened to a "tug-of-war", with activity promoting ER sheets poised against tubule promoting activity, particularly that of membrane curvature-inducing reticulons. The near disappearance of rER sheets and concomitant emergence of dense tubular tangles in Ire1 mutants suggests a runaway win for tubule promotion, potentially due to a failure of sheet-forming activities, an abnormal regulation of tubule formation, or a combination of both. Ribosomes promote ER sheets and it is possible the loss ribosomes in mutant R cells destabilizes rER. Alternately, abnormal, dense Rtnl1 accumulations seen in mutant R cells may reflect a cannibalization of ER sheets by misregulated Rtnl1 with a consequent reduction in Rh1 synthesis; misregulated Rtnl1 could also account for the loss of the SRC cortical ER network. The dynamic, stereotyped differentiation of ER morphology underlying Drosophila R cell differentiation presents a genetically accessible system to investigate how developmental programs shape ER (Xu, 2016).

Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. The Drosophila EHD ortholog, PAST1, has been shown to associate predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. The present study investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. These results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia (Dorot, 2017).

In the Past1 null mutant flies no external eye abnormalities were observed, but the development of photoreceptors R1, R6 and R7, as well as of cone cells, was abnormal. In flies overexpressing PAST1 in the eye external abnormalities were noted, as well as an abnormal number of photoreceptors, cone cells and IPCs. Defects in ommatidium development accompanied by no external abnormalities in the eye have been previously reported for other mutations. For example, the external eye of mutants in Muscleblind (Mbl), a regulator of alternative splicing, appear to be normal, yet tangential sections revealed that they harbored ommatidia defects. Another example involves Crumbs (Crb), a transmembrane protein which is essential for biogenesis of adherens junctions and for establishing apical-basal polarity in Drosophila epithelia by downregulating endocytosis of Notch and Delta during eye development. External morphology of mutant Crb (crb^S87-2) was reported to be normal. However, internal structure of adult Crb^S87-2 mutant ommatidia was found to be defective with shortened and bulkier rhabdomeres, often in contact with each other rather than being distinct as in wild type eyes (Dorot, 2017).

Interestingly in Past1 null flies, the ommatidia always contained at least one R7 photoreceptor, with some having two R7 photoreceptors with a concomitant loss of either R1 or R6 or both. These results imply that differentiation to R1 or R6 was abrogated and instead, at least one R7 photoreceptor was developed. No change in Boss staining was detected in R7 cells of eye discs from Past1 null mutants or from transgenic GFP-PAST1 flies, while in Past1 null mutants the ommatidia always contained at least four cone cells and some ommatidia had five cone cells (Dorot, 2017).

Previously work has shown that Past1 genetically interacts with Notch in the wing. The results of the present study indicated that Past1 plays a role during eye development, which is known to be regulated by Notch. Interestingly, endocytosis of Notch seemed abnormal in early-mid pupal mutant eyes. Thus, higher level of Notch staining was detected at the vicinity of the plasma membrane of mutant pigment cells, indicating abrogated endocytosis. This result implies abnormal internalization of Notch in the absence of PAST1 and hints to the possibility that PAST1 modulates internalization of Notch during eye development. (Dorot, 2017).

The results well fit the model suggested by Tomlinson (2011) for the development of photoreceptors R1/R6/R7 and cone cells. According to this model Notch overexpression in either R1 or R6 photoreceptors, leads to development of one of these cells into an extra R7 photoreceptor. Additionally, overexpression of Notch in R1 or R6 combined with an absence of Sev leads to the appearance of an extra cone cell. It is proposed that lack of PAST1 elevates Notch activation in R1 and R6 photoreceptors, which subsequently develop into R7 or into cone cells. It is hypothesized that PAST1 negatively regulates Notch signaling in R1 and R6. Since penetrance of Past1 mutation in the eye is low, PAST1 is, most probably, not a major regulator of Notch signaling, but fine-tunes it. Further experiments are needed to establish the role of PAST1 in endocytosis of Notch (Dorot, 2017).

Eyes of flies overexpressing PAST1 were rough with regions of fused ommatidia and exhibited extra IPCs. Mutations that abrogate the normal cell death process of IPCs have been shown to lead to rough eyes. More so, overexpression of Notch (N^FL or N^ICD) in the pupal retina led to appearance of extra IPCs. Therefore, it is assumed that the rough eyes phenotype in the PAST1 transgenic flies is due to abnormal PCD of IPCs (Dorot, 2017).

This study examined the ability of Garland cells overexpressing PAST1 to internalize fluorescently labeled Texas-Red avidin, as was examined for Past1 mutants. Garland cells overexpressing Past1 displayed attenuated endocytosis. Overexpression of PAST1 had a dominant negative effect, showing approximately 50-60% attenuation in endocytosis. A similar dominant negative effect has also been previously shown in HeLa cells overexpressing the mammalian EHD2, leading to attenuation of plasma membrane internalization (Dorot, 2017).

In conclusion, the results presented in this work highlight the importance of PAST1 for development of R1,R6 and R7 photoreceptors of the fly ommatidia (Dorot, 2017).

In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, this study found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain (Shimozono, 2019).

Given the role Connector eNhancer of KSR (CNK) plays in RTK-elicited Ras/MAPK signaling, mutations in loci encoding general components of this pathway in flies were recovered in a CNK C-terminal-dependent (CCT) screen. They correspond to Star [S; trafficking factor for the EGFR ligand Spitz, Egfr, daughter of sevenless (dos; Gab2 homolog), Son of sevenless (Sos; RasGEF), Ras85D, rl/mapk, ksr, and pointed (pnt; ETS domain transcription factor mediating MAPK activity). Mutations in two genes previously identified in RTK-dependent screens, but of unclear function, were also isolated. These are kismet [kis; chromatin remodeler] and multiple ankyrin repeats single KH domain [mask; putative RNA-binding protein (Baril, 2014).

Mutant alleles not identified in classical RTK/MAPK-dependent genetic screens, but for which a functional link to RTK signaling in flies or in other organisms had been established were also recovered. These include Btk family kinase at 29A [Btk29A; the single representative of Tec family kinases]; Delta (Dl); and three Rap1 pathway loci, PDZ-GEF, Rap1, and canoe (cno). Finally, mutations were isolated in four additional loci not previously reported to influence RTK/MAPK signaling: Pre-mRNA-processing factor 19 (Prp19), BREFeldin A sensitivity 1 (Bre1), NEM sensitive factor 2 (Nsf2), and second mitotic wave missing (swm) (Baril, 2014).

The Prp19 locus encodes a core spliceosome component. A specific role for these proteins has been unvailed in the Ras/MAPK pathway. Indeed, several splicing factors, including Prp19 and Prp8, were identified in a genome-wide RNAi screen in S2 cells for modulators of Ras-induced MAPK activation. Incidentally, a single mutant allele of Prp8 was also recovered in the CCT screen. Characterization of their implication in the pathway revealed that they specifically regulate MAPK protein levels by controlling the alternative splicing of selected introns of the mapk pre-mRNAs. It is thus likely that Prp19 alleles were recovered in the CCT screen because of their impact on endogenous MAPK levels during eye development (Baril, 2014).

The association that the last three genes (Bre1, Nsf2, and swm) might have with respect to CCT activity is less clear. Bre1 encodes a RING finger-containing E3 ligase mediating histone H2B monoubiquitination. This modification contributes to specific histone epigenetic changes such as histone H3K4 and H3K79 methylation that correlate with transcriptional activation. A role for Bre1 in Notch- and Wingless-dependent gene expression has been reported, but whether it acts similarly downstream of RTK signaling is not known. Given the concerted, yet distinct role Notch and EGFR signaling play in morphogenetic furrow progression and thereby in eye development, it could well be that, as for Dl, Bre1 was recovered primarily for its function in Notch signaling (Baril, 2014).

Nsf2 encodes an AAA ATPase involved in vesicular trafficking and synaptic vesicle release. Previous genetic studies have also associated this gene with Notch and Wingless signaling, and thus this could be the basis for the isolation of Nsf2 alleles. Alternatively, Nsf2 activity could be required in trafficking events directly involved in RTK signaling (Baril, 2014).

The swm gene [aka Su(Rux)2B encodes a novel protein that comprises a CCCH zinc finger and a RNA recognition motif. SWM localizes to the nucleus and was found to play multiple roles during Drosophila development, although its precise molecular function is not known. During eye development, Swm regulates the proliferation of undifferentiated cells by controlling their G1/S transition. In particular, third instar eye discs deprived of SWM activity are reduced in size and, as epitomized by the gene name, they lack the second mitotic wave, which corresponds to a row of cells located at a few cells distance posterior to the morphogenetic furrow that undergo a unique and synchronous round of cell division. This event increases the pool of uncommitted cells used for completing ommatidial assembly. Both Notch and EGFR signaling are essential for cell cycle progression of the uncommitted cells in the second mitotic wave, but act at distinct steps. Whether the swm alleles were recovered because of their impact on the second mitotic wave or for another role of SWM in differentiating cells remains to be investigated (Baril, 2014).

The ability of the CCT screen to identify mutations in three loci linked to Rap1 signaling strongly suggests a functional relationship between CNK and Rap1 activity. Yet, no evidence was found for physical association between CNK and Rap1 or PDZ-GEF, and thus the molecular underpinning of this relationship is currently not known. One possibility for their genetic interactions could be through their separate roles in RTK-mediated events. Rap1 signaling promotes adherens junction formation in differentiating photoreceptor cells, which contributes to their clustering. This phenomenon, in turn, is thought to enable the cells to respond to extracellular cues promoting differentiation. By lowering Rap1 activity, cohesive contacts between differentiating cells would be suboptimal and thereby would impede ommatidial assembly to some degree. In this scenario, the impact of CCT expression on photoreceptor cell differentiation would be exacerbated by heterozygous mutations in Rap1-signaling components as these would reduce the sensitivity of developing cells to differentiation cues (Baril, 2014).

Interestingly, it has been noted that loss of Rap1 activity does not prevent EGFR-induced MAPK activation per se, and thus Rap1 does not appear to work like Ras as a direct RAF activator. However, the data for this conclusion were based on small Rap1 mutant clones that were close or within the morphogenetic furrow. This work was extended by producing larger clones depleted in Rap1 or PDZ-GEF activity. These clones covered the zone where R7 cell commitment normally occurs. Markedly, it was found that reduced Rap1 signaling in this area considerably decreased MAPK activity as well as global pTyr levels and thereby mimicked the loss of RTK activity. Consistent with this, a strong impairment in R7 cell fate specification was observed (Baril, 2014).

In agreement with thes findings, Mavromatakis (2012) recently showed by genetic means that R7 cell fate specification had an absolute requirement in Rap1 activity. According to their model, R7 cell precursors sense higher Notch signaling owing to their position in the developing ommatidium, which at this stage is antagonistic to Ras/MAPK-mediated neuronal differentiation. To counteract Notch signaling, Mavromatakis (2012) proposed that presumptive R7 cells turn on two RTKs (EGFR and SEV) to produce higher MAPK activity. Intriguingly, their work suggested that Rap1 was required downstream of SEV, although they could not distinguish whether Rap1 acted through the canonical MAPK pathway or parallel to it. This was investigated and it was found Rap1 does not seem to work directly through the MAPK pathway since ectopic expression of Rap1V12 during eye development or in cultured S2 cells did not promote MAPK phosphorylation. Moreover, depletion of Rap1 or PDZ-GEF by RNAi in S2 cells had no consequence on MAPK activation induced by SEV or EGFR. Although the precise mechanism by which Rap1 influences signaling downstream of SEV remains to be delineated, the combined data suggest that Rap1 works at two distinct levels in SEV-mediated signaling, that is, upstream of SEV by modulating the apical localization of SEV and downstream of SEV by a mechanism that has yet to be characterized (Baril, 2014).

Adherens junctions form a belt-like microdomain that encircles epithelial cells apically and that play a major role in cell-cell adhesion, motility, and polarity. One of the core structural components of adherens junctions is Ecad, which is a transmembrane glycoprotein that forms Ca2+-dependent homophilic interactions between adjacent cells. The intracellular portion of Ecad is complexed to the catenins that, in turn, mediate linkage to the actomyosin cytoskeleton. Studies conducted over the past 10 years in both vertebrate and invertebrate organisms demonstrate the critical role that Rap1 signaling plays in modulating the connections of adherens junctions to the actomyosin network, which then influence cell-cell adhesion, cell shape, and cell migration (Baril, 2014).

Although the current data are consistent with this view, they also hint at a new role for Rap1 signaling that is to control apical domain formation in developing photoreceptor cells. Given that adherens junctions may act as physical barriers between apical and basolateral membrane compartments, the influence of Rap1 on adherens junction dynamics could represent the mechanism by which Rap1 exerts its effect on the apical domain compartment. A more exciting alternative would be that Rap1 activity directly controls the formation of the apical domain. Work conducted in fly embryos by Choi (2013) recently provided evidence supporting this model. Indeed, not only did that study find that Rap1 activity is essential for establishing the apico-basal polarity of cellularizing embryos, but their data also suggest that it has a direct impact on the apical localization of Bazooka, a member of the Par complex, which then orchestrates apical domain assembly. Whether Rap1 signaling has a direct influence on cell polarity during eye development is still unclear. Nonetheless, further characterization of the impact that Rap1 signaling has on apical domain formation/maintenance should reveal novel aspects by which cell compartmentalization is brought about and regulated as well as how it connects to downstream signaling events in epithelial cells (Baril, 2014).

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. This study demonstrates that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. It was further demonstrated that integrin localization changes dynamically during the rotation process. The data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90 degrees rotation movement (Thuveson, 2019).

Recent connectome analyses of the entire synaptic circuit in the nervous system have provided tremendous insights into how neural processing occurs through the synaptic relay of neural information. Conversely, the extent to which ephaptic transmission which does not depend on the synapses contributes to the relay of neural information, especially beyond a distance between adjacent neurons and to neural processing remains unclear. This study shows that ephaptic transmission mediated by extracellular potential changes in female Drosophila melanogaster can reach >200 μm, equivalent to the depth of its brain. Furthermore, ephaptic transmission driven by retinal photoreceptor cells mediates light-evoked firing rate increases in olfactory sensory neurons. These results indicate that ephaptic transmission contributes to sensory responses that can change momentarily in a context-dependent manner (Ikeda, 2022).

This study found that ephaptic transmission of light information from photoreceptor cells in the retina mediates the increase in firing rate in the olfactory sensory neurons (OSNs) during odor stimulations. This study has not revealed whether the ephaptic transmission directly changes the firing rate of the OSNs. Amputation of the antennal nerve abolished the firing rate increases during sustained light, suggesting that once the light information might be received by neurons in the brain, the information would be relayed by the neurons through the antennal nerve to the antenna, resulting in the firing rate increases in the OSNs (Ikeda, 2022).

While ephaptic coupling has been reported earlier, such as between neighboring neurons within the same sensillum, or between Purkinje cells, which is at a distance of <100 μm, this study shows that ephaptic transmission reaches >200 μm in vivo, equivalent to the depth of the entire fly brain, beyond the distance between neighboring neurons. Light stimulations cause -10 mV field potential deflections in a retina. If endogenous fields in the brain are neglected, light stimulations may induce ~33.3 mV/mm electric field between the retina and center of the brain (0 mV), since the distance between them is ~300 μm. This electric field is strong enough to modulate neural activities, as even weaker electric fields (<0.5 mV/mm) changed the firing patterns of neurons in vitro (Ikeda, 2022).

In rodents, the firing rate of cerebellar Purkinje cells either decreased or increased when a current was injected into the extracellular field around their axons, causing field potential changes of 0.2 mV. In insects, odor-evoked field potential oscillations whose amplitude is comparable with that caused by the current injection in the rodents, are induced by synchronous firing of olfactory neurons in the antennal lobe which are mediated by GABAergic neurons forming reciprocal synapses with excitatory projection neurons. Changes in the extracellular field potential are commonly observed in many nervous systems. While such extracellular field potential activities have been considered as a side effect of synchronized spiking of neurons, this study suggests that such field potential changes evoked by a sensory stimulus can control the excitability of distant neurons, in addition to adjacent neurons. As ephaptic transmission is more effective at a short distance, the ephaptic transmission from the retinae may contribute significantly to firing rate changes in downstream neurons of the photoreceptor cells in the optic lobe (Ikeda, 2022).

This study also revealed that odor responses of OSNs were clearly modulated when light conditions changed transiently. This mechanism may help flies switch attention to newly presented sensory cues or maintain attention toward those remaining after the change. Turning the light on, for example, reduces the firing rates of the OSNs, which may enable the flies to pay more attention to visual information, whereas turning the light off increases the firing rates of the OSNs, which may help them attend to olfactory sensory cues (Ikeda, 2022).

Recent connectome analyses have revealed the entire synaptic network in the CNS in Drosophila and provides insight into how neural information is subject to synaptic relays to determine the behavioral output. This study has shown that ephaptic relays also contribute to modulating the firing rate of distant neurons and modify the sensory responses that can change momentarily in a context-dependent manner should also be considered. To build an integrated model of the fly brain, ephaptic relay of neural information should be considered. The compound eye-antenna model would be a suitable model to determine the role of ephaptic transmission in neural processing (Ikeda, 2022).

Animals generally have either compound eyes, which have evolved repeatedly in different invertebrates, or camera eyes, which have evolved many times across the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. Only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led to speculation as to whether there are conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, an in-depth comparative whole-tissue transcriptomics approach was used. Specifically, the larval principal camera eyes were dissected into SupC- and retina-containing regions and the respective transcriptomes were generated. This analysis revealed several conserved features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate the extent of conservation, the transcriptomes were compared with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results provide molecular evidence for the deep conservation of SupCs in addition to photoreceptor cells, raising essential questions about the evolutionary origin of eye-specific glia in animals (Rathore, 2023).

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. This study explored the role of Drosophila PRL (dPRL) in photoreceptor cell development. dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na(+)/K(+)-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium (Chen, 2023).

During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORβ activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. This study shows that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORβ promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, a novel interaction was identified between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision (Bunker, 2023).

How complex three-dimensional (3D) organs coordinate cellular morphogenetic events to achieve the correct final form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. This study combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells coordinately organize tissue pattern to support retinal integrity. These experiments revealed an unanticipated intercellular feedback mechanism whereby correct cellular differentiation of either cell type can non-autonomously induce cytoskeletal remodeling in the other Abl mutant cell type, restoring retinal pattern and integrity. It is proposed that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis (Sun, 2023).

Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. This study profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. Both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, no correlation was found with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, these data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing (Jauregui-Lozano, 2023).

Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activity-dependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although this study has established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, RNAi screening was performed against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, the decreasing synapse number was directly quantified using a GFP-tagged presynaptic protein marker. Custom-made image analysis software was used, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl was used as a candidate gene responsible for synaptic assembly. Finally, a new model is presented of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly (Osaka, 2023).

Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. This study describes a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. These studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; these studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain (Tsai, 2019).

These studies demonstrate that disrupting the biosynthesis of specific membrane phospholipids causes adult-onset degeneration of R cells and loss of synaptic vesicles. These two phenotypes arise via distinct molecular mechanisms that can be doubly dissociated using genetic and physiological manipulations. Degeneration of the axon terminal is an activity-dependent process that requires calcium-mediated vesicle fusion. Conversely, loss of synaptic vesicles is driven by activation of the transcription factor SREBP. Thus, in these cells, SREBP is activated by alterations in the levels of specific phospholipids. Here, SREBP affects the expression of a specific subset of genes that are largely not directly involved in lipid regulation, thus defining a previously unknown SREBP function. Rather, SREBP activation leads to reduced expression of four tetraspanins. Restoring expression of either of two of these tetraspanins suppresses the effects of SREBP activation, demonstrating that tetraspanins are functional effectors of SREBP in photoreceptors. Thus, a specialized feedback loop from the synaptic terminal to the nucleus links the levels of specific phospholipids to photoreceptor function and synaptic vesicle number. It is proposed that this feedback loop matches the vesicular demand for phospholipids to their production. As SREBP is evolutionarily conserved, and recent studies have linked SREBP to neuronal damage in several contexts, it is speculated that this feedback loop plays a central role in maintaining synaptic vesicle pools in the healthy aging brain (Tsai, 2019).

In Drosophila, mutations that disrupt phospholipid biosynthesis cause broad defects in brain function, including increased seizure activity and photoreceptor degeneration. However, these and other studies examining phospholipid composition in flies have either not quantified phospholipid levels or have not differentiated different phospholipid species. These studies using a high-resolution lipidomic approach demonstrate that biosynthesis of specific PE and PC species is required for maintaining synaptic vesicle pools and the axon terminal in adult photoreceptors. Moreover, the biosynthetic enzyme Pect is found at the axon terminal. It is speculated that the production of specific phospholipids can occur locally, coupling precise levels of phospholipids to the cellular processes that require them in the axonal compartment. Finally, recent work has demonstrated that derivatives of very long chain (VLC) PC species are neuroprotective in vertebrate photoreceptors and neurons. Although this study detected only one VLC PC precursor, PC c44:12, representing 0.01% of the total PC species in the fly retina, future studies will determine the extent to which derivatives of this or other PC or PE species play roles in maintaining adult photoreceptor axons and synapses in Drosophila. This work suggests the following model. The ultrastructural analysis of pect mutants reveals phenotypes in the axon terminal that are strongly reminiscent of those in endocytic mutants. Consistent with this, blocking exocytosis in pect mutants by either reducing light exposure or by genetic means suppresses axon terminal degeneration. It is therefore inferred that the inability to retrieve synaptic vesicles from the plasma membrane is sufficient to cause neuronal degeneration and that the availability of specific phospholipids can be rate limiting for endocytosis. These results are consistent with previous studies in C. elegans that demonstrated that a phospholipid desaturase causes defects in endocytosis through effects on synaptojanin, a critical component in endocytosis. At the same time, altering phospholipid production may also impair vesicle biogenesis, in which case blocking synaptic transmission could suppress neuronal degeneration by removing the demand for vesicle biogenesis via an as-yet-unknown mechanism (Tsai, 2019).

SREBP is a central regulator of genes involved in lipid biosynthesis in many cell types. The current data support the notion that SREBP plays an additional role in Drosophila photoreceptors. As the levels of only a few phospholipids are altered in pect mutants, SREBP activation appears linked to the detection of changes in levels in these PE and PC species. Moreover, although activation of SREBP does upregulate a small number of genes involved in lipid biosynthesis, it also downregulates many genes involved in phototransduction and synaptic function. Among these, genetic interaction studies demonstrate that tetraspanins are functionally critical SREBP effectors. Tetraspanins are transmembrane proteins that have been linked to synapse development, lysosomal function in R cells, and to outer segment structure and function in the vertebrate retina. Moreover, recent work has demonstrated that they can serve as cholesterol-binding proteins, further implicating this family in the regulation of membrane function. Although unraveling the specific molecular mechanisms that link tetraspanin function to synaptic vesicle pools remains a challenge for future studies, the current model for this role of SREBP represents an extension of SREBP's long-standing role in regulating lipid biosynthesis. In particular, a central role for phospholipids that is unique to neurons is as a critical component of synaptic vesicles. It is hypothesized that, when SREBP is activated and tetraspanin expression is reduced, either the biogenesis of synaptic vesicles is downregulated or their turnover and degradation is increased, shrinking the synaptic vesicle pool in an activity-independent manner. As a result, the cellular demand for the specific phospholipids found in synaptic vesicles is reduced. More broadly, these studies suggest that SREBP might complement its long-standing role in lipid biosynthesis with an additional role in controlling phospholipid utilization. Finally, by combining the high-resolution lipidomic approach this study developed to work with small populations of labeled cells with the powerful genetic tools available in this system, future work may shed further light on the regulation of SREBP activity and phospholipid levels (Tsai, 2019).

SREBP has been linked to both neurodegenerative disease and stroke. Recent studies in flies have demonstrated that reactive oxygen species can activate SREBP to cause lipid droplet formation in glia. However, the molecular mechanisms by which SREBP might act in these contexts are unknown. In addition, mutations in a human tetraspanins have been linked to intellectual disability. The demonstration that SREBP acts through tetraspanins to regulate synaptic vesicle pools and negatively regulates other genes required for synaptic function suggests a unifying mechanism for these seemingly disparate observations. Taken together, these studies argue that SREBP plays an evolutionarily conserved role in regulating neuronal and synaptic function, suggesting a link between the neuronal phospholipid repertoire and synapse maintenance in the adult brain (Tsai, 2019).

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).

This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).

The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).

Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).

Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).

Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).

dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).

As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).

In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).

The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation, with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context. The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).

Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).

N-cad mutant ommatidia appear to over-rotate unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).

The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).

The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).

The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).

There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).

Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells. It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).

Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argos^rlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).

Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).

Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos, with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR. This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).

Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).

This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl^-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).

The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).

The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. An enhancer-trap screen was performed using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBac(AC109) and piggyBac(AC783) are expressed in R8 cells and piggyBac(AC887) is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. The expression pattern was demonstrated in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and it is proposed that these lines are useful tools for studying the development of the visual system (Terzioglu Kara, 2020).

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process genetic screens were performed using a collection of 480 P{EP} transposon insertion strains. Genes were identified in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3 percent in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes (Earl, 2020).

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. This study addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to understanding of apical growth during morphogenesis (Hebbar, 2020).

Despite the importance of intact microvilli for the homeostasis of many epithelia, regulators controlling microvilli formation are only partially understood. This study used the rhabdomere of Drosophila PRCs, a highly expanded and elaborated apical membrane, as a model to dissect mechanisms controlling the formation of microvilli. It was shown that increased transcription of fatty acid 2-hydroylase (fa2h), followed by increased levels of hydroxylated sphingolipids, is associated with defective rhabdomere morphogenesis. This is, at least partially, due to impaired apical trafficking of Rh1, an important structural component of the rhabdomeral membrane. These results also contribute to understanding of the role of crb in rhabdomere morphogenesis. They imply that increased levels of hydroxylated sphingolipids particularly impact one aspect of rhabdomeral growth, namely the addition of new microvilli, resulting in the extension of the rhabdomere along the proximodistal axis. Rhabdomeral thickness, determined by the length of the microvilli, is not obviously affected by changes in these lipids (Hebbar, 2020).

The connection between hydroxylated sphingolipid metabolism and apical domain morphogenesis is not unprecedented. Increased hydroxylation of sphingolipids was observed to be one of the major lipidomic changes during the polarization of MDCK cells, and inhibition of sphingolipid biosynthesis resulted in a reduced number of apical microvilli on their apical surface. Likewise, the Caenorhabditis elegans orthologue of fa2h, fath-1, was identified in a screen for genes affecting intestinal polarity. Until now, however, the link between hydroxylated sphingolipids and their metabolism and polarization of epithelial cells was limited to circumstantial evidence. This study now clearly demonstrate that manipulating hydroxylated sphingolipid metabolism, at least in a sensitized genetic background, is sufficient to modulate rhabdomere/apical domain extension (Hebbar, 2020).

How do hydroxylated sphingolipids and their metabolism affect rhabdomeric/apical domain growth? Given the low abundance of hydroxylated sphingolipids in the overall fly eye lipidome, it is considered unlikely that they play any major structural role in rhabdomere morphogenesis, e.g., by promoting the stability of Rhodopsin in the rhabdomeric membrane. The conclusion is favored that afa2h and thereby hydroxylated sphingolipids play a role as regulators of apical trafficking. This conclusion is based on two findings: (1) less newly synthesized Rh1 is delivered to the rhabdomeres upon overexpression of afa2h, as revealed by blue light-induced chromophore supply (BLICS) assays, which mimic Rh1 pulse-chase experiments; and (2) a similar defect in Rh1 trafficking has been observed in crb mutant PRCs , which have increased levels of afa2h expression. In particular, an effect on post-Golgi trafficking of Rh1 via the Rab11-mediated pathway was identified. Accumulation of cytoplasmic vesicles is observed upon genetic perturbation of Rab11 and/or genes that encode for Rab11 interacting proteins. However, no obvious accumulation of similar vesicles was observed in crb mutant PRCs. Thus, the trafficking defect observed upon afa2h overexpression cannot be attributed to a general increase/decrease in Rab11 compartments, but rather alludes to a defect in sorting apical cargo. These results are consistent with studies in C. elegans, where apical compartments labeled with Rab11 and Rab7 are affected upon loss of fath-1 (Li, 2018; Hebbar, 2020 and references therein).

Hydroxylated sphingolipids, as glycosphingolipids, are proposed to regulate the sorting of apically directed vesicles by combining different polarity cues (Zhang, 2011). In line with this, it is proposed that increased afa2h transcription (and hence an increase in hydroxylated sphingolipids) prevents apical membrane (rhabdomere) growth by inhibiting trafficking of Rh1 via the apical Rab11 compartments. By regulating the amount of Rh1 delivered to the rhabdomere, afa2h modulates the amount of rhabdomeric membrane in Drosophila PRCs, and hence has an impact on rhabdomere growth. Whether afa2h additionally promotes stability of Rhodopsin in the rhabdomeric membrane via the presence of more hydroxylated sphingolipids could not be addressed here. Interestingly, exogenously labeled hydroxylated fatty acids (the precursors of hydroxylated sphingolipids) preferentially distributed to apically localized membrane-bound compartments in the epithelial cells of the C. elegans gut (Li et al, 2018) (Hebbar, 2020).

Unexpectedly, afa2h overexpression or knockdown in an otherwise wild-type background does not result in an obvious rhabdomeric extension phenotypes. This may be because Rh1 trafficking depends on the concerted action of Rab1, Rab6, Rab11, Rip11, and MyoV as well as the Rab-effector proteins such as Parcas (Rab11GEF) and Rab11 interacting proteins such as dRip11, and to the amount of F-actin. Although sphingolipids can induce changes in the cytoskeleton, no overt changes in the staining of F-actin were observed in the rhabdomere. Therefore, it is speculated that increased afa2h acts to fine-tune growth by slowing down delivery of Rh1 and hence addition of new rhabdomeric membrane (Hebbar, 2020).

Yet, in a sensitized background, i.e., in cells homozygous mutant for crb, which have impaired Rh1 trafficking or upon carotenoid depletion, which also reduces Rh1 levels, afa2h overexpression now decreases Rh1 delivery via Rab11. Crb normally limits oxidative stress, and the resulting oxidative status (low oxidative stress) normally limits afa2h expression. In crb mutants, however, an altered oxidative status (increased oxidative stress) causes an up-regulation of afa2h transcription, which, in turn, results in severely reduced Rh1 delivery and improperly extended rhabdomeres. The afa2h dependence of rhabdomeric growth described in this study is only one aspect of the pleiotropic cellular response to an altered redox status of increased oxidative stress signaling due to loss of crb. Interestingly, loss of crb in epithelial cells of larval salivary glands impairs Rab6-, Rab11-, and Rab30-dependent apical trafficking and hence apical membrane homeostasis. Whether this is also caused by increased oxidative stress and/or defects in sphingolipid metabolism remains to be analyzed (Hebbar, 2020).

In conclusion, this work elucidates an interplay between oxidative stress, lipid metabolism, and apical domain growth during PRC morphogenesis. It will be interesting to investigate whether this link between cell metabolism and apical membrane growth and morphogenesis is a more widespread phenomenon in epithelial biology (Hebbar, 2020).

Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, a paradigm was used for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. This study found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. It is concluded that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation (Meserve, 2018).

Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. This study identified Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFβ) superfamily, that includes TGFβ and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, this study showed that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, a dynamic response of retinal cells was found to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFβ/BMP signaling, as supplementation of BMP4 or inhibition of TGFβ using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. These data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained (Kramer, 2021).

The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. This study integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When the Clock:Cycle complex was disrupted by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, changes were observed in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, expression of ClkDN in photoreceptors was shown to lead to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, these data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress (Jauregui-Lozano, 2022).

Expansion of the number of contiguous nucleotide repeats that normally exist within certain human genes is the cause of multiple human diseases. Earlier work has shown that expression of alleles containing nucleotide repeat expansions can be reduced differentially by inhibiting production of SUPT4H or SUPT5H, highly conserved cellular proteins that interact to form the transcription elongation complex, DSIF (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor). DSIF assists in the elongation of mRNA molecules by attaching to RNA polymerase II (RNAPII) via an SUPT5H binding site and forming a structural clamp that maintains RNAPII occupancy of template DNA as the polymerase proceeds along the template . A decrease in production or function of SUPT4H or SUPT5H has been found to decrease synthesis of transcripts encoded by genes containing nucleotide repeat expansions including HTT, the gene that causes Huntington's Disease, the C9orf72 locus associated with amyotrophic lateral sclerosis and frontotemporal dementia, and NOP56, the gene associated with spinocerebellar atrophy type 36 (SCA36), and it has been suggested that SUPT4H or SUPT5H may be a target for treatment of certain diseases caused by nucleotide repeat expansions. As interaction between SUPT4H and SUPT5H to form the DSIF complex is required for these proteins to form the structural clamp that maintains RNAPII on DNA template, this study sought to identify compounds that interfere with the SUPT4H-SUPT5H interaction and to elucidate their effects on mutant HTT gene products. This study describes the results of experiments aimed at: 1) identifying chemicals that can interfere with the SUPT4H/5H interaction, 2) determining whether chemical interference with the interaction recapitulates the effects of decreasing SUPT4H or SUPT5H on expression of genes containing expanded nucleotide repeats, and 3) determining whether chemical interference with the interaction has phenotypic effects (Deng, 2022).

Decreasing the expression of the SUPT4H or SUPT5H components of the DSIF complex can lower production of mRNAs encoded by mutant gene alleles containing nucleotide repeat expansions, and also can modify phenotypes associated with repeat expansions. These findings have led to proposals that that chemical or genetic targeting of SUPT4H or SUPT5H may be useful therapeutically. The results reported in this study indicate that chemical interference with the interaction of SUPT4H and SUPT5H is achievable, that such interference -which has been confirmed by two independent reporter assays and a direct biochemical assay-can lower the abundance of mutant HTT gene products in cultured cells and an HD animal model, and that chemical targeting of DSIF complex formation can mitigate phenotypic effects of repeat expansions. However, the broad and essential biochemical functions of DSIF, raise the prospect that therapeutic targeting of DSIF may be challenging. As SUPT4H and SUPT5H can act individually, as well as in complex with each other, the effects of targeting DSIF also may differ from the effects of targeting its individual components (Deng, 2022).

Compounds of multiple chemical classes potentially may interfere with the SUPT4H-SUPT5H interaction. Among the compounds identified by the screening assays was 6-azauridine, a previously studied nucleoside inhibitor of de novo uridine-5'-monophosphate productive pathway and consequently of nucleic acid synthesis and cell division. Addition of uridine to cell cultures reversed the effects of approximately equimolar amounts of 6-AZA on global nucleic acid synthesis without affecting mutant HTT expression, demonstrating the distinctness of these two effects of the compound (Deng, 2022).

Loss of medium spiny neurons (MSNs) in the striatum is a characteristic feature of HD and other neurodegenerative diseases. This study used CRISPR/Cas9 gene editing methodology to shorten the number of HTT gene CAG repeats in HD patient MSNs to a nonpathological length, and found that shortening of repeats in these congenic cells was associated with diminished sensitivity to H2O2 exposure. Treatment with 6-AZA partially reversed the incremental sensitivity of cells containing expanded repeats, but did not affect H2O2 sensitivity in cells containing shorter repeats (Deng, 2022).

Analogous partial reversal of phenotypic effects of mutant HTT expression was observed also in the adult Drosophila compound eye, which has been widely used as a model for Huntington's Disease and other human neurodegenerative disorders. No loss of Drosophila larval viability was detected at a 6-AZA concentration that rescued animals displaying the rough eye phenotype. However, the ability of uridine supplementation to reverse the global effects of 6-AZA on nucleic acid synthesis in cell culture raises the possibility that such supplementation may prove useful also in mammalian models during in vivo studies (Deng, 2022).

Whereas the pathogenic effects of repeat expansions in HD and certain other diseases have been observed most clearly in neuronal cells, they are also evident in non-CNS tissues. In the current experiments, they were observed in MSNs, in neuronal cells, in blood cells, and in photoreceptor cells of the eye-and in replicating and nonreplicating cells. Whereas chemical interference with the SUPT4H-SUPT5H interaction has the potential for affecting multiple tissues simultaneously, differences in the length of repeats as well as tissue-specific factors unrelated to DSIF may influence the results of such interference (Deng, 2022).

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. This study profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. This integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. It is proposed that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090 (Hall, 2021).

Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. This study used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Evidence is provided that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease (Periasamy, 2022).

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging (Hodge, 2022).

Progressive declines in circadian rhythms are one of the most common hallmarks of aging observed across most lifeforms. Quantifying the strength, or amplitude, of circadian rhythms is an accurate metric for predicting chronological age. Many cellular processes involved in aging (e.g., metabolism, cellular proliferation, DNA repair mechanisms, etc.) display robust cyclic activities. Both genetic and environmental disruptions to circadian rhythms are associated with accelerated aging and reduced longevity. These observations suggest that circadian rhythms may not merely be a biomarker of aging; rather, declines in circadian rhythms might play a causal role. The observation that DR and DR-memetics, such as calorie restriction and time-restricted feeding, improve biological rhythms suggests that clocks may play a fundamental role in mediating their lifespan-extending benefits (Kato, 2022).

This study identified circadian processes that are selectively amplified by DR. The findings demonstrate that DR amplifies circadian homeostatic processes in the eye, some of which are required for DR to delay visual senescence and improve longevity in Drosophila. Disrupting CLK function within photoreceptors accelerates visual declines and shortens lifespan, while overexpressing wild-type CLK protects against age-associated declines in vision and rescues AL-dependent declines in photoreceptor function. These data also demonstrate that photoreceptor stress has deleterious effects on organismal health; overstimulation of the photoreceptors induced a systemic immune response and reduced longevity (Kato, 2022).

Among the more interesting and unexpecting findings of this study is the observation that the Drosophila eye influences systemic immune responses, as elevated AMP expression was observed in the bodies of flies overexpressing CLK-Δ pan-neuronally and in flies with forced photoreceptor degeneration (ATPα-RNAi). It is possible that GAL4 misexpression may promote inflammatory responses in the fly bodies, although this study found a reduction in systemic inflammation in the rhodopsin-null lines suggesting that this phenomenon can originate at the photoreceptor. These systemic immune responses correlated with lifespan changes (increased body AMP expression is associated with declines in longevity and vice versa), similar to what is observed with chronic inflammation or “inflammaging” in other models. However, it cannot be concluded whether neuronal or eye-mediated increases in systemic inflammation are causal to aging in other tissues. Furthermore, the mechanisms by which the Drosophila eye, and, more specifically, the photoreceptor influence systemic immune responses are unclear. It is speculated that photoreceptor degeneration may disrupt the retinal-blood barrier such that damage signals from the eye propagate through to the hemolymph and activate AMP expression in distal tissues. Future studies are aimed at elucidating this mechanism, and its effect on longevity (Kato, 2022).

The findings of this study establish the eye as a diet-sensitive regulator of lifespan. DR's neuroprotective role in the photoreceptors appears to be mediated via the transcription factor CLK, which promotes the rhythmic oscillation of genes involved in the suppression of phototoxic cell stress. Given that CLK transcriptionally regulates circadian and non-circadian transcripts, future investigations may determine whether the time-of-day regulation of these genes by CLK is germane to promoting eye health with age. These studies may also examine whether the DR-mediated benefits on visual senescence and photoreceptor viability are mediated solely by CLK as a transcription factor (as demonstrated here) or whether circadian clock function (rhythmic output) is required. The findings also support the notion that age-related declines in the visual system impose a high cost on an organism's physiology. Perhaps this provides an alternative hypothesis for why several cave-dwelling animals, whose visual systems have undergone regressive evolution (e.g., cave-dwelling fish and naked-mole rats), are especially long-lived. Failing to develop a visual system may act as a pro-survival mechanism allowing organisms to avoid the damage and inflammation triggered by age-related retinal degeneration. Ultimately, developing a visual system, which is critical for reproduction and survival, may be detrimental to an organism later in life. Thus, vision may be an example of an antagonistically pleiotropic mechanism that shapes lifespan (Kato, 2022).

Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation (Nam, 2016).

Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. The Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless (Wernet, 2006).

The ability to discriminate between colours has evolved independently in vertebrates and invertebrates. However, despite the obvious differences in eye development and design, both flies and humans have developed retinal mosaics where classes of photoreceptor cells (PRs) with different spectral sensitivity are randomly distributed. The compound eye of Drosophila consists of ~800 optical units (ommatidia), each containing eight PRs in addition to accessory cells. In each ommatidium, the six 'outer PRs' (R1-R6) function like the vertebrate rod cells, as they are required for motion detection in dim light. These cells express the broad-spectrum rhodopsin, Rh1. The 'inner PRs' (R7 and R8) may be viewed as the equivalent of the colour-sensitive vertebrate cone cells, which express a range of different rhodopsin molecules (Wernet, 2006).

The general rule of sensory receptor exclusion also applies to Drosophila ommatidia, where only one rhodopsin gene is expressed by a given PR. The expression of inner PR rhodopsins can be used to distinguish three ommatidial subtypes. Two of the subtypes are distributed randomly throughout the retina: ~30% of ommatidia express ultraviolet-sensitive Rh3 in R7 cells and blue-sensitive Rh5 in R8 cells, and therefore are specialized in the detection of short wavelengths ('pale' ommatidia). The remaining ~70% express another ultraviolet-sensitive opsin (Rh4) in R7 and green-sensitive Rh6 in R8, making them more responsive to longer wavelengths ('yellow' ommatidia). The coupled expression of Rh3/Rh5 or Rh4/Rh6 within the same ommatidium results from communication between R7 and R8. In the dorsal rim area (DRA), a third type of ommatidia exists in which both R7 and R8 express ultraviolet-sensitive Rh3. These ommatidia are used to detect the e-vector of polarized sunlight for orientation. Spatially localized polarized light detectors and stochastically distributed colour-sensitive ommatidia therefore reflect two fundamentally different specification strategies that shape the retinal mosaic of Drosophila (Wernet, 2006).

The current model for specifying colour-sensitive ommatidia combines stochastic and instructive steps. First, a subset of R7 (pale R7, pR7) stochastically chooses Rh3 expression over the 'R7 default', Rh4. Second, these cells then impose the p fate (Rh5) onto R8 (pale R8, pR8) of the same ommatidium (Wernet, 2006).

This study reports the identification of spineless (ss) as a key regulatory gene for establishing the retinal mosaic. ss encodes the Drosophila homologue of the human arylhydrocarbon ('dioxin') receptor, a member of the bHLH-PAS (basic helix-loop-helix- Period-Arnt-Single-minded) family of transcription factors. At mid-pupation, ss is stochastically expressed in a majority of R7 that seem to correspond to the y subtype. ss is both necessary and sufficient to specify the yellow R7 (yR7) fate and subsequently the entire y ommatidia; pR7 cells are thus specified by default, and stochastic expression of ss represents the key regulatory event defining the retinal mosaic required for fly colour vision (Wernet, 2006).

homothorax (hth) has been identified as the key regulatory gene necessary and sufficient for the specification of DRA ommatidia. ss and hth cause similar homeotic phenotypes: that is, complete (hth) or partial (ss, 'aristapedia') transformation of antennae into legs. Therefore, a potential role of ss in ommatidial subtype specification was tested by generating whole-mutant eyes, as well as mitotic clones, lacking ss function using the null allele ss^D115.7 and the ey-FLP/FRT technique. Owing to ey-FLP expression in the antennal imaginal disc, ss mutant flies showed a strong aristapedia phenotyp, but lacked any obvious morphological eye phenotype. However, expression of rhodopsin genes was severely affected. In wild-type eyes, Rh3 is found in ~30% of R7 cells, as well as in both R7 and R8 of DRA-ommatidia, whereas the remaining ~70% of R7 contain Rh4. In ss mutant eyes, Rh4 was completely absent, whereas Rh3 was expanded into all R7 cells. The total number of ommatidia was not reduced, indicating that R7 cells were mis-specified into pR7, rather than yR7 being specifically eliminated. ss mutant mitotic clones were morphologically wild type; however, Rh3 was always present in mutant R7 cells (marked by the absence of ß-galactosidase (ß-gal) expression), whereas Rh4 was always lost (Wernet, 2006).

To test whether the R7 ss phenotype was cell autonomous, individual mutant R7 cells were generated using the MARCM technique. All mutant R7 cells [marked by the presence of green fluorescent protein (GFP) expression] contained Rh3 and never Rh4, demonstrating that ss is required cell autonomously in R7 to induce Rh4 expression. DRA ommatidia were correctly specified in ss mutant eyes, since Rh3 was expressed normally in both DRA R7 and R8 cells. Therefore, ss is necessary for the establishment of the yR7 subtype without affecting PR fate specification (Wernet, 2006).

The ommatidial subtypes are first specified in R7, which then instruct R8. Therefore, ss mutant eyes should exhibit a rhodopsin phenotype in R8. In wild types, ~30% of R8 cells contain Rh5, and the remaining ~70% contain Rh6. In ss mutant eyes, the large majority (up to 95%) of R8 contained Rh5, with some R8 still containing Rh6. However, most of these remaining yR8 cells were located in the dorsal third of the eye. In this part of the retina, instruction of pale R8 (pR8) by pR7 is less efficient, resulting in ommatidia with odd-coupled (Rh3/Rh6) rhodopsin expression. In ss mutants, the frequency of such ommatidia was significantly increased in the dorsal region. To test whether the R8 opsin phenotype of ss mutants resulted from the inability of some mutant R7 cells to properly instruct R8, rather than from ss being directly required in R8, sevenless; spineless (sev; ss) double-mutant eyes were generated (Wernet, 2006).

These eyes, which lacked R7 cells, always exhibited the sev single-mutant phenotype, with virtually all R8 cells containing Rh6. This indicates that ss is required in R7 for the formation of the yR7 subtype, and consequently for the formation of yR8, without being directly required in R8 PRs (Wernet, 2006).

Whether ss was also sufficient to induce the y ommatidial subtype was tested. Overexpression of ss in all developing PRs using a strong LGMR (long glass multiple reporter)-Gal4 driver and UAS-ss (LGMR.ss flies) resulted in a rough eye phenotype, as well as a dramatic rhodopsin phenotype: Rh4 was activated in all PRs throughout the eye (R1-R6 as well as R7 and R8), as revealed by ectopic expression of an Rh4-GFP reporter in many PRs per ommatidium compared with wild type. To avoid the strong phenotype in the eye, ss was misexpressed using the weaker, variegated GMR driver, sGMR (short GMR)-Gal4³¹ (sGMR.ss flies). This led to strong ectopic induction of Rh4 in many PRs without severely affecting retinal morphology. This ectopic induction of Rh4 was also observed in sev mutants, and was thus independent of R7. Rh3 was still detected in some R7 in sGMR.ss flies, presumably due to the lack of variegated Gal4 expression in these cells, whereas Rh4 was expanded to some outer PRs. However, co-localization of Rh3 and Rh4 was never observed, confirming that gain of Rh4 in R7 cells always leads to the exclusion of Rh3. In contrast, gain of Rh4 in outer PRs did not lead to the exclusion of Rh1; frequent coexpression of Rh1 and Rh4 was observed (Wernet, 2006).

Using an Rh4-lacZ reporter construct in LGMR.ss flies, it was found that ß-gal-positive PR axons projected to both lamina and medulla, confirming the expansion of Rh4 into outer PRs. However, Rh4-expressing outer PRs were not transformed into genuine R7 cells, since they maintained their lamina projections. Notably, DRA inner PRs were the only cells not expressing Rh4, suggesting that the DRA fate, specified by the gene hth, antagonizes ss function. Expression of Rh3 and Rh5 was completely lost (including in the DRA, where no rhodopsin was detected), while Rh6 expression was found in most R8 cells. This resulted in R8 coexpressing Rh4 and Rh6, demonstrating that the 'one sensory receptor per cell' rule can be broken in Drosophila PRs, as has been shown in other insects. Therefore, ectopic induction of the yR7 fate by ss specifically excludes the formation of pR7 cells. As a consequence, R8 cells expressing Rh5 are not induced, with most R8 expressing Rh6. Rh6 was never found in outer PRs, supporting the hypothesis that ss is required only in R7 for the choice between Rh3 and Rh4, and not directly in R8 for the Rh6 choice. In LGMR.ss flies, the specification of outer versus inner PRs (markers spalt and seven up) or of R7 versus R8 (prospero and senseless) was normal. Thus, ss acts by segregating ommatidial subtypes downstream of early PR specification events (Wernet, 2006).

Colour PR cell fate determination seems to be a late event in PR differentiation. To test whether ss can transform the R7 fate at late stages of development, the PanR7-Gal4 driver (which is also expressed in DRA R8 cells) was used. Late mis-expression of ss induced the y fate (Rh4) in all R7 cells, whereas Rh3 was absent. Opsin expression in the DRA was also altered, with Homothorax-positive cells (both R7 and R8) now expressing Rh4. Hence, it is possible to reprogramme the R7 fate at later stages of differentiation, as PanR7-Gal4 becomes activated at the time of rhodopsin expression. Surprisingly, expression of R8 rhodopsins outside the DRA was not affected, since the distribution of Rh5 and Rh6 resembled the wild type. As a result, many ommatidia manifested the very unusual coupling of Rh4 in R7 and Rh5 in R8. Therefore, although ss is able to reprogramme all R7 late in development, R8 cannot revert their fate once they have been instructed to become pR8, and they maintain Rh5. Two antagonistic genes expressed in either of the two R8 subtypes have been identified that act together as a molecular consolidation system responsible for this inertia of R8. To confirm that late expression of ss exclusively in R7 is sufficient to transform R7, ss was mis-expressed in ss^D115.7 mutants using PanR7-Gal4. This was sufficient to induce Rh4 and to repress Rh3. R8 were again not reprogrammed and exhibited the ss mutant phenotype, with many R8 cells expressing Rh5 (Wernet, 2006).

All of the results presented above strongly indicated that ss must be expressed in the y subtype of R7 at some point during pupal development. Since several attempts to generate an anti-Spineless antibody had failed, in situ hybridization was used to detect ss messenger RNA in the retina at mid-pupation. At ~50% pupation, ss mRNA was detected in four neuronal cells per ommatidial cluster, one PR and three bristle cells. The PR was also labelled by anti-Prospero, confirming its identity as R7. Although the expression levels of ss in bristle cells seemed uniformly high, levels of ss expression varied considerably among R7 cells, ranging from very faint to very strong in 60%-80% of R7. A 1.6 kilobase 'eye enhancer' fragment (ss_eye) was also identified within the ss promoter that drives PR-specific expression. After crossing ss eye-Gal4 to UAS-ß-gal::NLS (nuclear localization sequence) reporters, PR-specific ss expression was first detected at mid-pupation-that is, approximately one day before rhodopsins are expressed, and before any visible molecular or morphological distinction between ommatidial subtypes. A single PR per ommatidium, which was identified as R7 through co-staining with Prospero, expressed ss. Thus, the ss eye enhancer recapitulates endogenous ss expression in PRs. ss expression was detected in 60%-80% of R7, correlating well with the distribution of Rh4 in adult retina. Like Rh4-expressing ommatidia, ß-gal-positive ommatidia were more abundant in the dorsal half of the eye, and no ß-gal expression was detected in the DRA (marked by Homothorax), where Rh4 is also never expressed. ss eye-Gal4 expression was detectable for only ~2 h at midpupation (Wernet, 2006).

Although it was not possible to directly co-stain for ss and Rh4 (which starts to be expressed one day later during pupation), it seems that at mid-pupation a short pulse of ss is deployed in a large subset of R7, which will become yR7 (Wernet, 2006).

Whether a short pulse of ectopic ss expression was able to modify the entire retinal mosaic was tested using a heat shock-Gal4 driver (hs-Gal4) to temporally control ss expression (hs.ss flies). A 30-min heat shock at ~50% pupation indeed resulted in an increase of Rh4 expression with a concomitant reduction of Rh3 in adults. The phenotype varied extensively, from only R7 cells expressing Rh4 (~25% of the flies analysed had Rh4 in most R7), to almost every PR expressing Rh4. In contrast, a 30-min pulse of ss in one-day-old adult flies had no effect. Heat shocks during larval or early pupal stages were lethal. Thus, PRs are extremely sensitive to a short pulse of ss during mid-pupation, at the time when endogenous ss is normally expressed. To further study the mechanism of the stochastic choice between p and y ommatidia, the retinal mosaic was examined in different mutant backgrounds. Flies heterozygous for ss^D115.7 had fewer Rh4-expressing R7 cells. Since the ss^D115.7 allele affects only the ss coding sequence, heterozygous flies have two functional promoters, only one of which produces a functional protein, suggesting that the non-productive promoter might sequester limiting factor(s) that regulate(s) the expression levels of ss. If this hypothesis is correct, addition of extra copies of the ss promoter should have a similar effect. Indeed, the addition of two functional copies of the ss eye enhancer (ss eye-Gal4) in an otherwise wild-type background also caused a significant reduction of the yR7 subtype. Therefore, the level of Spineless expression is important for the induction of the yR7 fate, which is less efficient in cells where the amount of Spineless is reduced (Wernet, 2006).

Retinal patterning in Drosophila reveals an original mechanism for how PR mosaics can be generated: stochastic expression of a single transcription factor (Spineless) acts as a binary switch that transforms the seemingly homogeneous compound eye into a mosaic, distinguishing p and y subtypes. However, subtype specification and rhodopsin expression can be separated, since ss expression in yR7 has ceased well before the time of rhodopsin expression. Additional factors are therefore required downstream of ss to ensure expression of adult p- and y-specific markers such as rhodopsins and additional screening pigments. A revised two-step model is proposed for the stochastic specification of p and y ommatidia. First, R7 are stochastically divided into two subtypes by the induction of ss in yR7. ss-positive R7 express Rh4, whereas the remaining R7 choose the pR7 fate and express Rh3 by default. Second, only those R7 cells that did not express ss (pR7) retain the ability to induce the pR8 fate (Rh5), whereas yR8 express Rh6 by default. The 'default states' of R7 (Rh3) and R8 (Rh6) therefore belong to opposite subtypes. Expression of R8 rhodopsin genes is maintained by a bistable regulatory loop containing the genes warts and melted. Notably, the localized specification of polarization-sensitive DRA ommatidia by hth antagonizes the stochastic choice executed by ss, placing these two genes into a new regulatory relationship during retinal patterning. Therefore, the role of the transcription factor Spineless is to generate the retinal mosaic required for fly colour vision by distinguishing yR7 from pR7 cell fates, and preventing R7 from instructing the underlying R8 cells. Mosaic expression of sensory receptors has been described in detail for the olfactory system of both vertebrates and insects, and random PR mosaics have been described for humans and amphibians, as well as insects. Two transcription factors have been shown to regulate the specification of blue versus red/green cone cell fates in mammals. Upon mutation of either - the human nuclear receptor NR2E3 (also known as PNR) or the rodent thyroid hormone ß2 receptor - the number of blue cones is dramatically increased at the expense of green cones, leading to 'enhanced S-cone syndrome'. It should be noted that this retinal phenotype bears important similarity to the altered ommatidial mosaic in Drosophila ss mutants, where long wavelength-sensitive y ommatidia are lost at the expense of the short wavelength-sensitive p type (Wernet, 2006).

The stochastic cell fate choice occurs at the level of the ss promoter: the very short pulse of ss expression at mid-pupation is not only controlled temporally, but its levels are also critical, and only ~70% of R7 receive enough Spineless to commit to the yR7 fate. Elucidating the mechanism that controls ss expression will shed some light into the fascinating process of stochastic gene expression, and the identification of its downstream targets will provide insights into consolidation and maintenance of cell fates (Wernet, 2006).

Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). This study investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. The ss locus is in a compact state in undifferentiated cells. An early enhancer drives transcription in all R7 precursors, and the locus opens. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In Ss(ON) R7s, ss is open and competent for activation by a late enhancer, whereas in Ss(OFF) R7s, ss is compact, and repression prevents expression. These results suggest that a temporally dynamic antagonism, in which transcription drives large-scale decompaction and then compaction represses transcription, controls stochastic fate specification (Voortman, 2022).

Cell fate specification is controlled by lineage, signaling, and stochastic regulatory inputs, leading to highly precise developmental outcomes. Stochastic mechanisms promote diversity in populations of photoreceptors (PRs), olfactory neurons, motor neurons, and immune cells. Despite the importance of stochastic cell fate specification, how cells randomly choose between fates is poorly understood (Voortman, 2022).

Stochastic cell fate specification is best understood in prokaryotes. One well-characterized example is the bet-hedging mechanism utilized by Bacillus subtilis. To minimize losses in a changing environment, populations of genetically identical bacteria maintain a subpopulation of cells that are competent for DNA uptake. The transient and random transition into the competent fate is controlled by expression of the transcriptional regulator ComK. Though most cells maintain low expression of ComK, a subset will experience a pulse of ComK expression that exceeds a threshold and induces a transition to the competent fate. A similar mechanism occurs in the HIV life cycle, where transcription of the regulatory factor trans-activator of transcription (Tat) determines the switch from proviral latency to active replication. Thus, stochastic cell fate specification often requires a pulse of expression of a critical regulator that determines a fate decision (Voortman, 2022).

In addition to transcriptional dynamics, chromatin-mediated repression is a key mechanism mediating stochastic fate specification. In mice, each olfactory sensory neuron (OSN) expresses only one olfactory receptor (OR) gene from a battery of ∼1,300 possibilities. Despite residing in numerous clusters across many chromosomes, all ∼1,300 OR genes are repressed and coalesce into heterochromatic foci within the nucleus prior to OR selection. In mutants that impact chromatin modifications and nuclear organization, co-expression of multiple ORs is observed. While the mechanism of selection remains elusive, a single OR allele escapes the repressive heterochromatic environment and is expressed in each OSN. Thus, chromatin-mediated silencing and selective de-silencing are paramount for the stochastic expression of a single OR gene (Voortman, 2022).

The random mosaic of R7 PRs in the fly eye provides a paradigm to study the integration of transcription and chromatin-mediated repression in stochastic cell fate specification. In the fly eye, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) establishes the random pattern of two R7 subtypes across the retina. SsON R7s express Rhodopsin 4 (Rh4), while SsOFF R7s express Rhodopsin 3 (Rh3). In wild-type flies, each R7 has a 67% chance of adopting the SsON R7 fate and a 33% chance of assuming the SsOFF R7 fate, yielding a consistent ratio yet unique, random pattern of R7 subtypes across eyes. In ss protein null mutants, all R7s express Rh3. The stochastic ON/OFF ss expression is controlled by an enhancer (late enhancer, LE) that drives expression in all R7s and silencers that limit expression to a subset of R7s (Voortman, 2022).

This study describes a mechanism that controls stochastic R7 subtype specification. Initially, the ss locus is compact in all undifferentiated cells. An early enhancer (EE) drives ss expression and the ss locus opens in all R7 precursors during larval development. Expression ceases and the ss locus randomly compacts or remains open. In R7s in which ss remains open, the LE drives ss expression and SsON R7 fate. In R7s with compact chromatin, repression prevents expression driven by the LE, yielding the SsOFF R7 fate. The data suggest that stochastic fate specification is controlled by the dynamic, intertwined relationship of transcription and chromatin: transcription opens chromatin then chromatin compaction represses transcription. Transcription was found to be a source of stochasticity as modulating early transcription in precursors alters the proportions of alternative R7 fates (Voortman, 2022).

This study investigated how regulation of transcription and chromatin compaction at the ss locus controls stochastic R7 patterning in the fly eye. ss is initially in a compact, repressed state in undifferentiated cells. This compacted state is similar in other SsOFF cell types including peripodial cells and SsOFF R7s. As the eye develops, ss is transcribed in precursors and chromatin is opened. ss transcription and large-scale decompaction are lost in mutants deleting the EE or the promoter, suggesting that ss transcription drives the opening of the ss locus in precursors early (Voortman, 2022).

As the cells mature, ss expression ceases, and the ss locus compacts during the transition from the precursor to the differentiating cell phase. Our observations that (1) the ss locus is open in SsON R7s and compact in SsOFF R7s and (2) the similarity of median compaction in differentiating cells and all R7s (including SsON R7s and SsOFF R7s) suggest that the ss locus assumes either an open or compact state in differentiating cells that is maintained until terminal R7 subtype specification. Our data are consistent with stable compaction states in differentiating cells, but they cannot rule out changes during this phase of R7 development (Voortman, 2022).

ss expression and compaction during the transition from precursor to differentiating cell phases are critical processes that determine the stochastic R7 fate choice. Decreasing EE activity reduced ss expression in precursors and the proportion of SsON R7s. Extending ss transcription into the differentiating cell phase increased the proportion of SsON R7s. It is proposed that variable activation and duration of transcription in each precursor determines the probability of recompaction, which ultimately dictates the SsON or SsOFF expression state in R7s (Voortman, 2022).

In the last stage of R7 subtype specification, ss expression driven by the LE is repressed in a subset of R7s. The repression reporter strategy showed that repression at the ss locus limits expression to a subset of R7s. The chromatin compaction assays showed that the ss locus is open in SsON R7s and compact in SsOFF R7s. Deletion of the LE ablated expression but did not alter compaction in R7s. Thus, the chromatin state is set and maintained independent of expression at this stage of R7 maturation. Further, open chromatin is not sufficient to activate ss expression. Together, these data suggest that open chromatin allows activation by the LE whereas compact chromatin represses ss expression (Voortman, 2022).

Based on these findings, a mechanism is proposed that controls stochastic R7 subtype specification. The ss locus is in a compact state in undifferentiated cells. The EE drives transcription and opens the ss locus in precursors. Early expression ceases and the ss locus randomly assumes an open or compact state in differentiating cells. R7s with open chromatin at the ss locus reactivate ss and take on the SsON R7 fate, whereas R7s with compact chromatin at the ss locus repress ss and take on the SsOFF R7 fate (Voortman, 2022).

A key aspect of this mechanism is the initial 'priming' or opening of the ss locus during the early expression in precursors. Transcription-based priming plays important roles in several stereotyped developmental programs. A well-understood example has been described in C. elegans, where the bilateral pair of ASE gustatory neurons display asymmetric gene expression and function. Stereotyped specification of the left neuron ASEL is dependent upon the asymmetric expression of the microRNA lsy-6, achieved by a 'prime and boost' mechanism. Several cell divisions prior to the birth of the terminal ASEL neuron, a pulse of lsy-6 expression in the precursor cell promotes decompaction of the lsy-6 locus. This decompacted state is maintained in the ASEL lineage throughout development, allowing for reactivation of lsy-6 in the terminal ASEL neuron. In the ASER lineage that never experiences the early pulse of lsy-6 expression, the locus remains in a repressed, compacted state, preventing later activation by transcription factors that are expressed in both ASE neurons. Thus, early transcription of a key regulator (lsy-6) promotes one cell fate (ASEL) by antagonizing chromatin-mediated repression important for the specification of the alternative fate (ASER) (Voortman, 2022).

The transcription-based prime and boost mechanism controlling ASEL/R sensory neuron specification in C. elegans has many similarities to the mechanism that this study has identified for R7 subtype specification. In both systems, early expression of a key regulator in precursor cells opens a locus (prime) so that it can be reactivated later upon terminal specification (boost). A major difference is that the ASEL/R decision requires priming in only the ASEL lineage to reproducibly generate the ASEL fate, whereas the R7 subtype decision utilizes priming in all precursors, which opens the chromatin followed by variable chromatin compaction and repression that ultimately determines the SsON or SsOFF R7 fate (Voortman, 2022).

Both the ASEL/R and R7 subtype decisions also exhibit a window of inactivity between the early and late expression phases. However, this window appears to play two very different roles. In the ASEL/R decision in worms, the early priming of the lsy-6 locus occurs several cell divisions prior to terminal differentiation. The time between the prime and boost is an obstacle that must be overcome to remember the early developmental event. In contrast, the window between the early and late stages of ss expression appears to enable chromatin compaction and repression that determine the SsON or SsOFF expression states in R7s (Voortman, 2022).

Though stochastic fate specification is an important feature of many cell fate programs, general features of these mechanisms have not been identified. In the bacterium Bacillus subtilus, transcriptional regulation is critical, as ComK transcription drives a stochastic cell fate switch to the 'competent' fate. In both the competence decision in bacteria and R7 subtype specification in flies, all 'precursor' cells express the key regulator, yet only a subset undergo the cell fate switch (Voortman, 2022).

Stochastic R7 subtype specification in flies also shares mechanistic features with OR selection in mice, particularly in the repression of alternative fates. In the olfactory system, OR genes are found in a compact heterochromatic region in the nucleus, with one gene that escapes repression and activates. Similarly, chromatin compaction and repression play key roles in determining ssON and ssOFF R7 fates. The current studies in flies bridge the roles of transcription in bacteria and chromatin in mice for stochastic cell fate specification (Voortman, 2022).

Understanding of the relationship between transcription and chromatin is often a chicken and egg problem: it is unclear whether transcription state dictates large-scale chromatin state or vice versa. This study provides evidence that clearly identifies these cause-effect relationships and show how they change during development. The EE drives transcription to open chromatin in precursors. In differentiating cells, the EE ceases to function and transcription stops. Chromatin remains open or closes, marking the stochastic step. Finally, the LE turns on in mature R7s. In cells where the locus is open, transcription reinitiates, while in cells where the locus is closed, transcription is repressed. Thus, initially, transcription state regulates chromatin state and later, chromatin state controls transcription state. Our studies not only outline this simple mechanism, but also identify how the stochastic step is regulated. The stochastic step occurs as cells cease ss transcription in the precursor phase and assume the open or compact chromatin state in differentiating cells. Decreasing or extending early transcription alters the probability of chromatin closing and ultimately, the proportion of R7 subtypes. Thus, variability in the duration of early transcription is likely a key input that determines the stochastic decision. These findings provide an important step in understanding how transcription and large-scale chromatin states regulate one another to control how cells randomly assume fates (Voortman, 2022).

The data suggest that transcription drives large-scale chromatin decompaction and then compaction represses transcription, which controls stochastic cell fate specification. This study focused on large-scale chromatin remodeling. It is hypothesized that local changes of histone modifications at the enhancer and promoter likely precede transcription in precursors. The heterogeneity of the tissue limited testing this hypothesis in a cell-type-specific manner in intact tissue. Moreover, it is concluded that chromatin compaction represses ss expression in a subset of R7s. The repression reporter experiments showed that the chromatin context at the ss locus is sufficient to repress expression in a subset of R7s. The DNA FISH experiments showed that the ss locus is open in ssON R7s and compact in ssOFF R7s. These experiments were limited as it was not possible to identify conditions to artificially induce compaction and show sufficiency of compaction to repress ss. Additionally, these studies were conducted in fixed tissue, limiting the observation of the rapid temporal interplay between transcription and chromatin. Future studies could address this challenge with live imaging, enabling assessment of the transcriptional and chromatin dynamics of the ss gene throughout the maturation of individual R7s during subtype specification (Voortman, 2022).

Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. This study investigated how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein despite expressing both proteins with pulsatile dynamics. A rapid and sustained two-fold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a two-fold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how two-fold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program (Bernasek, 2023).

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. This study reports that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, it was found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1(H890A) homozygous mosaic eyes. Furthermore, the Ire1(H890A) mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. These results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation (Mitra, 2021).

Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. It has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. To distinguish between these two models, the expression patterns were analyzed of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. It was found that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. This combinatorial regulatory logic is compared to the regulatory logic of olfactory receptor genes and potential implications for the evolution of rhodopsins is discussed (Poupault, 2021).

Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. This study discovered a novel phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require inositol 1,4,5-triphosphate receptor (IP3R), suggesting these waves are mediated by Ca2+ releases from ER stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitating stress fiber contraction and floor morphogenesis. Further, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating ECM and its interaction with stress fibers in eye morphogenesis. It is proposed that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor (Ready, 2021).

Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. This study investigated the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. This study finds that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven (Gallagher, 2022).

Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. This study shows that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc (Ren, 2022).

Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. This study used developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, this study showed that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors (Kiral, 2021).

The phenomenon of tissue fluidity-cells' ability to rearrange relative to each other in confluent tissues-has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. This study utilized in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, this study demonstrated that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. This study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening molecular understanding of tissue fluidity (Founounou, 2021).

Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized Drosophila photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 Drosophila ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat. This study investigated the function of Strat in fly photoreceptors and found that polarized transport in both the basolateral and rhabdomere membrane domains was inhibited in Strat-deficient photoreceptors. 79% and 55% reductions in Rab10 and Rab35 levels, respectively, were also observed but no reduction in Rab11 levels in whole-eye homozygous clones of Strat(null). Moreover, Rab35 was localized in the rhabdomere, and loss of Rab35 resulted in impaired Rh1 transport to the rhabdomere. These results indicate that Strat is essential for the stable expression of Rab10 and Rab35, which regulate basolateral and rhabdomere transport, respectively, in fly photoreceptors (Ochi, 2022).

Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pk^sple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. By characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, this study showed that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pk^sple and pk^pk can enforce robust R3/R4 fate decisions, but only pk^sple can correctly orient them along the dorsal-ventral axis. In contrast, pk^sple and pk^pk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. It is proposed that expression dynamics and competitive interactions determine isoform participation in these processes. It is proposed that the selective requirement for pk^sple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation (Cho, 2022).

The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. This study reports that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components β-Catenin (β-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in β-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of βPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of β-Cat. It is thus proposed that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology (DeSantis, 2023).

Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, this study showed Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. The light-dependent regulation of Pkc53E was demonstrated and it was demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCβ4/NorpA as degeneration of norpA(P24) photoreceptors was enhanced by a reduced Pkc53E activity. It was further shown that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton (Shieh, 2023).

The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. Single-cell RNA sequencing was performed to elucidate the cause of dedifferentiation and to determine the fate of these cells. Dedifferentiated cells were found to adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. This work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation (Rader, 2023).

The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1-R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision (see Normalized spectral quantum sensitivity of the different rhodopsins in the photoreceptor subtypes R7p, R7y, R8p, R8y, and R1-R6). This study investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a 'UV vs. blue' choice, flies with only R1-R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a 'blue vs. green' choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1-R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1-R6 (Yamaguchi, 2010).

Phototaxis consists of at least three behavioral components: (1) detection of an object or a light source in visual space; (2) the attractiveness or aversiveness of the light stimulus at this location; and (3) a motor output (goal-directed walking and turning). Genetic dissection of the visual input to phototaxis relies on the assumption that phototaxis stays intact even if some of these inputs are deleted, i.e., that the mutants can still detect the locations of the two stimuli and move toward or away from them. Although in mutants affecting the R1-R6 subsystem, walking speed, turning, and orientation toward stationary objects are affected, these behaviors are sufficiently intact to support phototaxis. In the experiments reported in this study, light intensities were chosen such that all mutants had at least one photoreceptor type that could detect the light sources and guide walking and turning behavior. This allowed measuring of the second component, the differential attractiveness of the two stimuli, which determined the choice between the two monochromatic lights depending upon the photoreceptor types available in the respective fly strains. Flies could judge the attractiveness of a light source on the basis of its spectral composition and/or spectrally weighted brightness. In the following paragraphs the contributions of the four types of photoreceptors R1-R6, R7, R8p, and R8y to the attractiveness function are discussed (Yamaguchi, 2010).

Blocking or removing any one of the photoreceptor types shifts the preference away from the point of neutrality in at least one of the choice tests. Silencing the R1-R6 cells leaves the flies with a modest blue preference in the UV/B choice, and replacing their broadly sensitive opsin Rh1 by the UV-sensitive opsin Rh3 shifts the preference in the B/G choice to blue. Removing or silencing R7 in the presence of R1-R6 shifts the preference in the UV/B choice to blue and in the B/G choice to green. Inactivating the green-sensitive Rh6 opsin in R8y cells shifts the preference in the B/G choice to blue and in the UV/B choice to UV. Inactivating blue-sensitive Rh5 in the R8p cells causes a green preference in the B/G choice and a UV preference in the UV/B choice (Yamaguchi, 2010).

Each of the three photoreceptor subsystems (R1-R6, R7, R8) alone can drive phototaxis. Flies with only photoreceptors R1-R6 (sev rh5² rh6¹ triple mutants) show a blue preference in the UV/B choice, implying that this subsystem not only mediates optomotor responses and orientation but also can mediate the attractiveness function in phototaxis, i.e., that, with only R1-R6 photoreceptors, flies compare the quantum flux of two light sources 180° apart (Yamaguchi, 2010).

The R8 subsystem alone can also mediate the attractiveness function in phototaxis (sev ninaE¹⁷), as had been shown before for the sev rdgB double mutant. The sev ninaE¹⁷ flies have a pronounced green preference in the B/G choice test, consistent with the absorption spectrum of Rh6 expressed in all R8 photoreceptors (Yamaguchi, 2010).

Flies that have only R7 photoreceptors operating (ninaE¹⁷, rh5², rh6¹) show phototaxis. Surprisingly, they have a very strong preference for blue in both choice tests. This could be explained if R7p inhibit R7y photoreceptors. Otherwise, the flies would show a strong UV preference in the UV/B choice. As an alternative explanation, however, it is possible that phototaxis is mediated by the ocelli in flies with only R7 photoreceptors (Yamaguchi, 2010).

As long as only one of the central photoreceptor subsystems R7 or R8 is inactivated, the results can be explained by a model in which the respective photoreceptors contribute roughly additively to the attractiveness function. In the honeybee, all three subtypes of photoreceptors (UV, blue, and green) also feed into phototaxis. Spectral mixing experiments in phototaxis are consistent with simple summation of quantal fluxes, and the action spectra of phototactic behavior also suggest pooling of all three photoreceptor types. Color information, i.e., comparison between different photoreceptors, does not appear to be used in honeybee phototaxis (Yamaguchi, 2010 and references therein).

A simple summation model thus can no longer explain the results of all of the genetic manipulations of photoreceptor types in Drosophila presented in this study. When all four photoreceptor types are intact, UV light is more attractive than would be expected from the sum of the UV attractiveness values of the two isolated major subsystems, R1-R6 and R7/R8. In the UV/B choice test that is balanced for wild type, both flies with only the R1-R6 subsystem (sev rh5² rh6¹) and flies with only the R7/R8 subsystem (rh1 > shi^ts or ninaE¹⁷) show a blue preference. It is thus necessary to postulate that an interaction between the two major retinal subsystems R1-R6 and R7/R8 enhances UV attractiveness. The interaction cannot be explained by an attenuation of the attractiveness of blue because no increased blue preference of the two mutants is observed in the B/G choice. The interaction can unambiguously be traced to the R7 cells. In fact, the strength of the contribution of R7 to the attractiveness function in the UV/B choice depends upon which of the other photoreceptor types are functional. No effect of R7 on spectral preference can be detected without a functional R1-R6 subsystem (comparing ninaE¹⁷ and sev ninaE¹⁷ in both choice tests. A moderate effect is found in the presence of R1-R6 and R8 (comparing wild type and sev; whereas the effect of R7 is large in the absence of the R8 subsystem (comparing rh5² rh6¹ and sev rh5² rh6¹ in UV/B choice (Yamaguchi, 2010).

The most parsimonious explanation of the data is to assume that, in the presence of a functional R8 subsystem, neither the R1-R6 nor the R7 subsystems on their own have a direct input to the attractiveness function. In the absence of R7 (mutant sev), the R1-R6 subsystem seems not to matter for attractiveness although the R8 subsystem is lacking spectral sensitivity in the UV. Likewise, in the absence of a functional R1-R6 subsystem, the R7 subsystem seems not to contribute. Interestingly, as soon as R8y photoreceptors (or R8p and R8y) are inactivated, the other two subsystems exert their influence on the attractiveness function independently of each other. A full model of these interactions would require a more complete investigation (Yamaguchi, 2010).

R1-R6 rhabdomeres degenerate in ninaE¹⁷ mutants, and this degeneration may have secondary effects on the R7/R8 cells. However, there is no significant difference between ninaE¹⁷ mutants and flies expressing rh1>shi^ts, in which R1-R6 function is disrupted without affecting rhabdomere structures. Moreover, it was recently reported that R8y are functional in ninaE¹⁷ mutants because circadian entrainment to red light, which is still observed in ninaE¹⁷, is abolished in ninaE¹⁷ rh6¹ double mutants (Yamaguchi, 2010).

The data reveal a further deviation from a simple summation of photoreceptor inputs to the attractiveness function. In the melt^GOF mutant, green-sensitive R8y cells are transformed into blue-sensitive R8p cells. This should shift their preference in the UV/B choice to blue. Yet, a UV shift is observed. In contrast, in the absence of R7 cells, the transformation of R8y to R8p photoreceptors (comparison between sev and sev melt^GOF) increases the blue preference in the UV/B choice as would be expected from spectral sensitivities. This might indicate that R8p cells enhance the input of the UV channel (R1-R6 × R7) to the attractiveness function. These findings, however, should be treated with caution as unknown developmental defects in the sev and melt^GOF mutants might account for the phenotypes (Yamaguchi, 2010).

Using a different phototaxis paradigm, Jacob (1977) proposed a model to explain the nonadditive effects observed in his phototaxis experiments. An inhibition of R1-R6 by R7/R8 was postulated and it was suggested that R7 cells function only when the R1-R6 system is intact. The current results ask for revision of this model. No general inhibition of the R1-R6 receptor subsystem by R7/R8 was found. The data suggest that neither the R1-R6 nor the R7 subsystems have access to the attractiveness function on their own, except in the absence of functional R8y photoreceptors. Moreover, R8p cells in the current tests had an effect only in the presence of functional R8y cells (Yamaguchi, 2010).

Rh3-expressing R8 cells in the dorsal rim area account for about 10% of all Rh3 expressing cells. These R8 are still present in sev flies, but are switched off in panR7> shi^ts flies. The comparison of these two lines does not reveal an effect of the Rh3-expressing R8 cells in the UV/B choice. Taking these cells into consideration therefore does not change any of the above conclusions (Yamaguchi, 2010).

This study has shown that all four photoreceptor types [R1-R6, R7(p, y), R8p, and R8y] are involved in phototaxis, in contrast to motion detection, which relies exclusively on R1-R6 photoreceptors. The wild-type attractiveness function cannot be described as the sum of the attractiveness functions of flies lacking one or more functional photoreceptor types. A multiplicative interaction is observed between photoreceptors R1-R6 and R7. In the absence of functional R1-R6 or R7, the attractiveness function is governed by R8. Only in the absence of R8 and one of the other two subsystems does the remaining subsystem govern the attractiveness function. A recent study on the neuronal substrate of spectral preference identified postsynaptic interneurons in the medulla (Gao, 2008) that are good candidates for mediating some of these interactions. Their involvement in differential phototaxis can now be tested (Yamaguchi, 2010).

The attractiveness function of differential phototaxis is easy to record, robust, and sensitive (for example, Rh5-expressing blue-sensitive cells account for only ~4% of all photoreceptors, yet yield a significant phenotype when they are not functional). Differential phototaxis may lend itself to mutant screens affecting other chromaticity computations in the brain, including color vision, at the circuit level (Yamaguchi, 2010).

Linearly polarized light originates from atmospheric scattering or surface reflections and is perceived by insects, spiders, cephalopods, crustaceans, and some vertebrates. Thus, the neural basis underlying how this fundamental quality of light is detected is of broad interest. Morphologically unique, polarization-sensitive ommatidia exist in the dorsal periphery of many insect retinas, forming the dorsal rim area (DRA). However, much less is known about the retinal substrates of behavioral responses to polarized reflections. Drosophila exhibits polarotactic behavior, spontaneously aligning with the e-vector of linearly polarized light, when stimuli are presented either dorsally or ventrally. By combining behavioral experiments with genetic dissection and ultrastructural analyses, it was shown that distinct photoreceptors mediate the two behaviors: inner photoreceptors R7+R8 of DRA ommatidia are necessary and sufficient for dorsal polarotaxis, whereas ventral responses are mediated by combinations of outer and inner photoreceptors, both of which manifest previously unknown features that render them polarization sensitive. It is concluded that Drosophila uses separate retinal pathways for the detection of linearly polarized light emanating from the sky or from shiny surfaces. This work establishes a behavioral paradigm that will enable genetic dissection of the circuits underlying polarization vision (Warnet, 2012).

This study defines the retinal substrates for both dorsal and ventral polarization vision in Drosophila. The DRA is necessary and sufficient for dorsal polarotactic responses, a result that strengthens studies in other insects, concluding that this region mediates responses to celestial polarized light. In addition, this work defines the retinal substrate for responses to ventral polarotactic stimuli, as would occur naturally by reflections from shiny surfaces like water or leaves. This work resolves the differences between previous behavioral studies of polarotactic behavior in flies by demonstrating that flies possess separate detectors to respond to distinct wavelengths, and sources, of polarized light (Warnet, 2012).

A ventral POL region has previously been described in the backswimmer Notonecta, which uses polarized reflections to locate water bodies. In this insect, inner photoreceptors in a small ventral region form orthogonal analyzer pairs with untwisted rhabdomeres much like a DRA. Drosophila uses a different strategy by exploiting the fact that photoreceptors with moderate or weak twist still provide enough PS to serve as polarization analyzers. In this way, other visual senses, such as the detection of motion and spectral cues, should be affected only minimally by the polarization of light Hence, unlike Notonecta with its specialized ventral retina, the generalist Drosophila incorporates ventral POL detectors while preserving other critical visual capacities (Warnet, 2012).

An interesting feature of this design is that different classes of photoreceptors form ventral POL analyzers depending on stimulus wavelength. Two subtypes are named 'pale' (p) and 'yellow' (y) and are randomly distributed across the retina. R7 cells each express one of two UV opsins rh3 (R7p), or rh4 (R7y), whereas the underlying R8 cells express either rh5 (R8p) or rh6 (R8y). Due to this chromatic heterogeneity, inner photoreceptors are thought to mediate color vision. In the UV range, R7p cells are necessary for normal polarotactic responses; correspondingly, ventral R7 cells are described with moderate to high estimated PS. However, sufficiency experiments also revealed the involvement of outer photoreceptors in polarization vision. Whereas R1-R3 appear to be weakly polarization sensitive, R4, R5, and possibly R6 show pronounced estimated PS due to reduced rhabdomeric twist. These results are consistent with intracellular recordings in Calliphora describing two classes of R1-R6, one of which retains some PS, even in the UV (Warnet, 2012).

Whereas R4 to R6 with their pronounced PS provide a basis for ventral polarotaxis via the outer photoreceptors, the contribution of R8 is less clear. It was found that R8 rhabdomeres twist strongly and, thus, R8 cells are expected to have low PS. In contrast, behavioral tests demonstrate that R8 can rescue polarotaxis in norpA mutants. Consistent with this, rare cases were found of very short R8 rhabdomeres exhibiting small twist ranges and correspondingly high expected PS. However, we cannot exclude the possibility that the apparent behavioral contributions of R8 could reflect low-level expression of the driver lines in R1-R6 (Warnet, 2012).

In larger flies, R7y and R8y as well as R1-R6 contain a UV-sensitizing pigment. Because this molecule is not covalently linked to the opsin protein, its function is independent of microvillar orientation, thereby diminishing PS in the UV range. In addition, these cells contain a C40 carotinoid, which both gives them their yellow appearance and induces anomalous dichroism, which further reduces PS. This may explain why R7p, but not R7y, can mediate ventral UV polarotaxis in Drosophila. The data describe an unexpected new role for 'pale' ommatidia outside the DRA (Warnet, 2012).

Moreover, the behavioral data confirm that in R1-R6 the UV-sensitizing pigment does not completely eliminate PS in the UV, as was previously reported. The contributions of the outer photoreceptors therefore become more pronounced when polarized green light is presented. That cellular contributions to ventral POL vision differ as a function of wavelength is particularly interesting because reflections from leaves contain much less UV (and more green light) than reflections from water. Hence, activation of distinct combinations of photoreceptors might convey specific meanings to the fly (Warnet, 2012).

The combination of polarization-sensitive outer and inner photoreceptors represents a new analyzer design, differing from those described in the DRA and the ventral retina of Notonecta. In particular, morphological data does not reveal an orthogonal organization of ventral analyzers. However, comparison between these channels might still increase quality and robustness of the signal. Nothing is known about the subtype-specific connectivity of R7p/R8p and their postsynaptic partners, and no electrophysiological data on polarization-opponent interneurons, or 'compass neurons', exist in flies. By establishing Drosophila as a model of polarization vision, these studies will enable genetic screens using quantitative behavioral assays to allow a complete dissection of the neural circuits involved in responding to this fundamental quality of light (Warnet, 2012).

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. This study shows that interplay of two transcription factors Senseless (Sens) and homeodomain transcription factor Hazy [PvuII-PstI homology 13, Pph13] control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. These findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner (Mishra, 2013).

In the larval eye, determination of primary or secondary precursors to acquire either Rh5-PR or Rh6-PR identity depends on the transcription factors Sal, Svp and Otd. Primary as well as secondary precursors have the developmental potential to express Rh5 or Rh6. During differentiation, a pulsed expression of Sens acts as a trigger to initiate a distinct developmental program: Sens acts genetically in a feedforward loop to inhibit the Rh6-PR cell-fate determinant Svp and to promote the Rh5-PR cell-fate determinant Sal. Similarly, in the adult retina, differentiation of 'inner' PRs R7 and R8 requires sens and sal. Sal is necessary for Sens expression in R8-PRs and misexpression of Sal is sufficient to induce Sens expression in the 'outer' PRs R1-R6 (Mishra, 2013).

Svp is exclusively expressed in R3/R4 and R1/R6 pairs of the outer PRs in early retina development. Initially, Sal is expressed in the R3/R4 PRs in order to promote Svp expression. Later, Svp represses Sal in R3/R4 PRs in order to prevent the transformation of R3/R4 into R7. Similarly in larval PRs Svp is repressing Sal in secondary precursors (Mishra, 2013).

Intriguingly, in R8 development in the adult retina Sens also provides two temporally separable functions: First, during R8 specification, lack of Sens in precursors results in a transformation of the cell into R2/R5 fate; second, during differentiation, Sens counteracts Pros to inhibit R7 cell fate and promotes R8 cell fate. Thus, Sens is an early genetic switch in R8-PRs and larval Rh5-PRs that represses an alternate cell fate (Mishra, 2013).

The lack of Sens results in a larval eye composed of only Rh6-PRs. Thus, the default state for both primary and secondary precursors is to differentiate into Rh6-expressing PRs. Rh6 is also the default state in adult R8 PRs: In the absence of R7 PRs (e.g. sevenless mutants) that send a signal to a subset of underlying R8 PRs, the majority of R8 PRs express Rh6. Thus, the genetic pathway initiated by the Sens pulse ensures that primary precursors choose a distinct developmental pathway by repressing the Rh6 ground state. The mechanisms that initiate and control this pulse of Sens remain to be discovered (Mishra, 2013).

In larval PRs as well as in the formation of sensory organ precursors (SOP) in the wing, Sens functions as a binary switch between two alternative cell fates. In the larval eye, this switch occurs when Sens is expressed in one cell type and not in the other. However, during wing disc development the cell fate choice in SOP formation is controlled by the levels, and not the presence or absence of Sens expression: high levels of Sens act synergistically with proneural genes to promote a neuronal fate, while in neighboring cells, low levels of Sens repress proneural gene expression, thereby promoting a non-SOP fate. Thus, Sens uses distinct molecular mechanisms to act as a switch between Rh5 versus Rh6-PR cell fate and SOP versus non-SOP cell fate (Mishra, 2013).

Transcription factors regulate developmental programs in a context- dependent fashion. An example is Sens, which has distinct functions in BO and eyelet development. First, during embryonic development, Sens acts as a key cell fate determinant by regulating transcription factors controlling PR-subtype specification. Second, during metamorphosis Sens inhibits ecdysone-induced apoptotic cell death. Third, in the adult eyelet Sens promotes Rh6 expression. Interestingly, the pro-survival function of Sens appears to be a conserved feature of Sens in other tissues and also in other animal species. In the salivary gland of Drosophila, Sens acts also as a survival factor of the salivary gland cells under the control of the bHLH transcription factor Sage. pag-3, a C.elegans homolog of Sens is involved in touch neuron gene expression and coordinated movement (Jia, 1996; Jia, 1997). Pag-3 was shown to act as a cell-survival factor in the ventral nerve cord and involved in the neuroblast cell fate and may affect neuronal differentiation of certain interneurons and motorneurons. In mice, Gfi1 is expressed in many neuronal precursors and differentiating neurons during embryonic development and is required for proper differentiation and maintenance of inner ear hair cells. Gfi1 mutant mice lose all cochlear hair cells through apoptosis, suggesting that its loss causes programmed cell death (Wallis, 2003). Taken together, these findings support that Sens and its orthologs function in cell fate determination and cell differentiation both in nervous system formation, but also play an essential role in the suppression of apoptosis (Mishra, 2013).

Hazy plays distinct roles in larval PRs and during metamorphosis. First, Hazy is essential during embryogenesis for proper PR differentiation. This early function of Hazy is essential for PRs to differentiate properly during embryogenesis, to express Rhodopsins and to subsequently maintain Rhodopsin expression during larval stages. This function of Hazy is similar to its role in rhabdomere formation in adult PRs and subsequent promotion of Rh6 expression, although it is not required for Rh5 in the adult retina. It is likely that Hazy exerts this function by binding to the RCSI site of the rhodopsin promoters, as has been suggested for the adult retin. Second, during metamorphosis Hazy is required in Rh6-PRs to repress sens, thus allowing these cells to undergo apoptosis. This highlights the reuse of a small number of TFs for distinct functions in the same cell type at distinct time points of PR development. How these temporally distinct developmental programs are controlled on a molecular level remains unresolved. It seems likely that the competence of the cell to respond to a specific transcription factor changes during development (Mishra, 2013).

rh5 and rh6 are expressed in different PRs at different developmental stages: rh5 is expressed in the larval eye and in the adult retina, whereas rh6 is expressed in the larval eye, the adult eyelet and the adult retina. However, the gene regulatory networks controlling rhodopsin expression are distinct in these organs. In the adult retina, a bi-stable feedback loop of the growth regulator melted and the tumor suppressor warts acts to specify Rh5 versus Rh6 cell fate, respectively, while in the larva, Sens, Sal, Svp and Otd control Rh5 versus Rh6 identity whereas Hazy has been shown to maintain Rhodopsin expression. A third genetic program acts downstream of EcR during metamorphosis in Rh5-PRs to switch to Rh6, which requires Sens (Mishra, 2013).

An intriguing question is how the developmental pathways to specify Rh5- or Rh6-cell fates converge on the regulatory sequences of these two genes. It seems likely that parts of the regulatory machinery acting on the rh5 and rh6 promoters are shared between the larval eye, adult retina and eyelet, especially as short minimal promoters are functional in all three different contexts. Future experiments will show how the activity of the identified trans-acting factors is integrated on these promoters to yield context-specific outcomes (Mishra, 2013).

Animals from worms and insects to birds and mammals show distinct body plans; however, the embryonic development of diverse body plans with tissues and organs within is controlled by a surprisingly few signaling pathways. It is well recognized that combinatorial use of and dynamic interactions among signaling pathways follow specific logic to control complex and accurate developmental signaling and patterning, but it remains elusive what such logic is, or even, what it looks like. This study developed a computational model for Drosophila eye development with innovated methods to reveal how interactions among multiple pathways control the dynamically generated hexagonal array of R8 cells. Two novel findings were obtained. First, the coupling between the long-range inductive signals produced by the proneural Hedgehog signaling and the short-range restrictive signals produced by the antineural Notch and EGFR signaling is essential for generating accurately spaced R8s. Second, the spatiotemporal orders of key signaling events reveal a robust pattern of lateral inhibition conducted by Atonal-coordinated Notch and EGFR signaling to collectively determine R8 patterning. This pattern, stipulating the orders of signaling and comparable to the protocols of communication, may help decipher the well-appreciated but poorly defined logic of developmental signaling (Zhu, 2016).

In the last larval instar, uncommitted progenitor cells in the Drosophila eye primordium start to adopt individual retinal cell fates, arrest their growth and proliferation, and initiate terminal differentiation into photoreceptor neurons and other retinal cell types. To explore the regulation of these processes, mRNA-Seq studies of the larval eye and antennal primordial were performed at multiple developmental stages. A total of 10,893 fly genes were expressed during these stages and could be adaptively clustered into gene groups, some of whose expression increases or decreases in parallel with the cessation of proliferation and onset of differentiation. Using in situ hybridization of a sample of 98 genes to verify spatial and temporal expression patterns, it is estimated that 534 genes or more are transcriptionally upregulated during retinal differentiation, and 1367 or more downregulated as progenitor cells differentiate. Each group of co-expressed genes is enriched for regulatory motifs recognized by co-expressed transcription factors, suggesting that they represent coherent transcriptional regulatory programs. Using available mutant strains, novel roles are described for the transcription factors SoxNeuro (SoxN), H6-like homeobox (Hmx), CG10253, without children (woc), Structure specific recognition protein (Ssrp), and multisex combs (mxc) (Quiquand, 2021).

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. This study shows that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, dac was demonstrated to enhance Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis (Bras-Pereira, 2016).

Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response (Kumar, 2015).

This paper used the Drosophila eye as a model system with which to study how morphogen gradients can be converted into sharply constrained tissue patterns. The action of the Wg morphogen gradient was examined and it was asked how the highest threshold response, the death of the peripheral ommatidia, is orchestrated. Three observations argue that the secondary Wg expressed by the cone cells combines with the primary Wg from the head capsule to generate a sufficient concentration to kill the ommatidia. First, when the Wg pathway is activated in all cone cells (pros->arm* were arm* is an N-terminally truncated form of Armadillo, a constitutive, cell autonomous activator of the Wg transduction pathway) there is an extended zone of apoptosis in the region where the primary Wg source is known to be high. Second, when the secondary Wg (that secreted by the cone cells) is removed the extended band of ommatidial death is lost. Third, when a level of Wg equivalent to that normally found in the peripheral regions is supplied to pros-arm* eyes all ommatidia now die. Thus, this represents a novel gradient read-out mechanism in which the primary morphogen (Wg derived from the head capsule) elicits a secondary morphogen expression (Wg expressed by the cone cells) in the target cells. Thereafter, the two sources unite to generate the high local morphogen concentration needed to direct the appropriate cell behaviors at that position (Kumar, 2015).

If there is a permissive zone in the periphery (∼3 ommatidial rows) in which the ommatidia will die if cone cell Wg expression occurs, then this raises the question of how the cone cells responses are normally tightly restricted to the peripheral-most row of ommatidia to ensure that only these ommatidia die. The following describes 1he mechanisms likely responsible for this restriction (Kumar, 2015).

(1) The high threshold of the ommatidial response: It is surmised that the cone cells have a high threshold response to the morphogen, and the initial responses to the primary Wg source (diffusing from the head capsule) is restricted to the outermost ommatidia. However, it can be envisioned that the secondary Wg secreted by the outer cone cells could diffuse and elicit the same output in the next ommatidial row, and an extreme view could see a relay mechanism in which even more internal rows of ommatidia could express Wg in their cone cells (Kumar, 2015).

(2) The role played by Notum:
The expression of Notum is similar to Snail family transcription factors in that it is expressed in the cone cells and 2°/3° PCs of the outermost ommatidia, and since Notum functions to inhibit the free diffusion of Wg, it likely acts to prevent Wg diffusion into more interior ommatidia. Indeed previous studies have shown that in notum mutant clones the zone of death expanded out into more interior rows. Thus Notum (and other mechanisms for preventing Wg diffusion) is seen as playing a critical role in restricting the ommatidial death to the outermost row of ommatidia (Kumar, 2015).

(3) Combining the high threshold response with the restriction of Wg diffusion: Consider the primary Wg diffusing from the head capsule. It enters the outer row of ommatidia and is of sufficient concentration to elicit the appropriate responses (the various expressions in the cone cells and 2°/3° PCs) but not at a level high enough to kill the ommatidia. The cone cells of the outermost row now begin to secrete the secondary Wg, but the concomitant expression of Notum by the cone cells and 2°/3° PCs of these ommatidia provide a barrier to the movement of both the primary and secondary sources of Wg. This restriction of Wg movement not only protects the more internal ommatidia, but ensures that the high levels of morphogen are constrained in the outermost ommatidia to provide the requisite signal for apoptosis (Kumar, 2015).

In addition to uncovering the synergy between the Wg derived from the head capsule and the cone cells, a number of phenomena relating to the behavior of the various cell types have been detected (Kumar, 2015).

(1) The early cone cell death: Following the collapse of the cone cells, the ommatidial apoptosis program begins with the death of cone cells themselves, followed ∼two hours later by the other ommatidial cells. This precocious cone cell death may represent a lower apoptosis threshold for these cells, but it is noted that they are sources of Wg secretion and likely experience autocrine and paracrine (between cone cells of the same ommatidium) Wg signaling as they collapse, and as such are more likely to reach the critical Wg activation level before the other cells (Kumar, 2015).

(2) The cone cell immunity to death: In pros-arm* eyes, in which all cone cell nuclei fall to the photoreceptor layer and express Wg, there is a wide swath of extended death at the periphery in which all cells of the ommatidia die (including the cone cells). But upon prevention of the cone cell nuclear fall by the expression of esg RNAi, the cone cells survive while the photoreceptors in the extended peripheral zone still die. In these ommatidia, levels of Wg needed to drive apoptosis are achieved, but the cone cells appear invulnerable to it. Whether this invulnerability results from the absence of Snail family transcription factors needed to prime the cone cells for the death signal, or whether by remaining in the apical location they somehow avoid the full level of Wg exposure remains unclear (Kumar, 2015).

(3) The fall of the cone cell nuclei: The maintenance of cone cell cell-bodies in the appropriate apical location is seemingly critical for the ommatidial stability and integrity, as their fall leads to the disruption of corneal lens units and delamination of photoreceptors. This fall appears to be directed by their expression of Snail family transcription factors. In pros-arm* eyes, the expression of esg.RNAi prevents the fall, and correspondingly the ectopic expression of esg in otherwise wild type cone cells engenders their nuclear fall (albeit prematurely). It was asked whether the fall of the cone cell nuclei resulted from a wholesale collapse of the apical junctions of the cone cells, but D/E-cadherin staining showed a normal apical junction pattern many hours after the nuclei had migrated basally. Thus it does not appear that the cone cells nuclei move basally because the cells lose their apical attachments, rather it is inferred that expression of the Snail family transcription factors reprograms some other behaviors of the cone cells. Such a behavior could be a switch in cell-type affinity. If cone cells normally maintain an apical location by adhesive differences with the photoreceptors, and if these adhesive differences are switched, then cone cell plasma membranes will then preferentially move to the photoreceptor layer. Since the nucleus defines the site of maximum cell body profile with corresponding maximum membrane area, then the fall of the nuclei may simply result from the cone cells acquiring an adhesive affinity with the photoreceptors. Other mechanisms can also be envisaged, in which, for example, motor machinery of the cell is used to reposition the cone cell nuclei in the more basal location (Kumar, 2015).

An appropriate Gal4 driver line is not available to activate gene expression selectively in the 1° PCs, and the mechanism of their death remains unresolved. In GMR.wg eyes, their death was observed coincident with the photoreceptors (following the apoptosis of the cone cells) and it is surmised that it is the high level of Wg derived from head capsule and the cone cells that directs their death. However, there are a number of indications from that offer clues to a more nuanced understanding of their behavior. Initially the nuclei of the 1° PCs flank the clustered photoreceptor nuclei in their more apical region, but when the cone cell nuclei fall, those of the 1° PCs are shunted more basally. This movement deeper into the photoreceptor layer may play a role in their death. A similar argument can be made from the analysis of * eyes in which 1° PCs are lost, but when Snail family transcription factors are removed from this background, the cone cell nuclei do not fall, and the 1° PCs do not die. Hence the 1° PCs behave in a similar manner as the cone cells; if their position is maintained they do not die even though ambient Wg concentrations are sufficient for their death. This may indicate a general principle; that cells need to be in the correct topological position to experience the death signal (Kumar, 2015).

Furthermore, in * eyes, the cone cell nuclear fall is accompanied by the loss of the 1° PCs even though the cone cells themselves do not die. The removal of Wg expression from the pros-arm* cone cells rescues the 1° PCs indicating that their loss is normally triggered by the cone cell Wg expression, and it is suspected that the low-level apoptosis seen in the main body of pros-arm* eyes may represent the death of the 1° PCs. If this is the case, then this suggests that the 1° PCs have a lower threshold Wg response for their apoptosis than the cone cells and photoreceptors (Kumar, 2015).

The death of the photoreceptors appears to simply require the additive of effects of the two Wg sources to trigger their death. But another feature has emerged from these studies – the idea that chronic exposure to sub-lethal levels of Wg triggers photoreceptor degeneration. Consider pros-arm*/esgRNAi eyes; here the photoreceptor death occurs only at the widened zone of peripheral apoptosis, but in the main body of the adult eyes ommatidia show degenerate rhabdomere-like tissue in the apical retinas. The presence of rhabdomere-like tissue suggests the differentiation and subsequent degeneration of the photoreceptors leaving them alive but in a runtish condition. Since this phenomenon is Wg dependent (it is absent when wgRNAi is additionally included) it is inferred that the persistent Wg expression from the cone cells chronically signals to the photoreceptors. Indeed, when GMR.wg/GMR.P35 eyes (in which the apoptosis mechanism is suppressed and the photoreceptors are therefore subject to chronic Wg exposure), were examined a similar degenerate phenotype occurred. This observation suggests another function for the removal of the outer-most row of ommatidia: if they were not removed, chronic exposure to high levels of Wg emanating from the head capsule would lead them to deteriorate into a runtish condition (Kumar, 2015).

A striking feature of the peripheral patterning mechanism is the timing aspect. The peripheral ommatidia are exposed to head capsule-derived Wg from the time of their birth. And yet they only respond to this Wg signal at defined times. The first occurs shortly after pupation when ac/da transcription is repressed and hth expression is induced. This corresponds with the surge in ecdysone expression that occurs in the animals at this time. The second response is the death of ommatidia at 42 h APF and this mechanism is closely tied with the large peak of ecdysone expression that occurs in the second day of pupation. Thus, it is speculated that Wg provides the spatial signal for peripheral patterning, but that the hormone system of the fly provides the temporal cue that determines when the spatial information can be utilized (Kumar, 2015).

It is concluded that the periphery of the fly eye is an excellent model system with which to study how morphogen gradients are decoded into discrete tissue types, and this study has delved into the mechanism that precisely restricts the spatial positioning of one of those tissue types. An intricate mechanism has been uncovered in which initial threshold responses lead to the local boosting of the morphogen signal while at the same time upregulating mechanisms to prevent the spread of the morphogen. Evidence is also provided to support the idea that appropriate spatial, temporal and topological context is required for the peripheral ommatidia to undergo developmental apoptosis (Kumar, 2015).

The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-alpha and IFN-beta inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway (Jenny, 2016).

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).

This study examined the cis-regulatory mechanisms that distinguish restricted from broad expression patterns for Rhodopsins and downstream phototransduction factors, respectively. All Rhs share the conserved Rhodopsin Core Sequence I (RCSI), which resembles the palindromic P3 motif (TAATYNRATTA), an optimal binding site for paired-class homeodomain proteins. Almost all known broadly expressed phototransduction genes contain a P3 motif in their proximal promoter. The presence of a conserved P3/RCSI motif within 100 base pairs (bps) of the Rh transcription start site (TSS) is significantly associated with enrichment in adult eyes. P3/RCSI is required for activation in photoreceptors because its mutation caused either a loss or a strong reduction in expression of 16 broad or restricted reporters, with the exception of Arr1. Moreover, expression of 10 out of 15 reporters was lost in mutants for the photoreceptor-specific transcription factor Pph13, a paired-class homeodomain protein that binds P3 and the Rh6 RCSI in vitro (Rister, 2015).

Because each Rh promoter has a highly conserved RCSI variant, the sufficiency of P3 and RCSI were tested to determine the significance of the specific differences between perfectly palindromic (P3) and imperfect motifs (RCSI). Four copies of the P3 motif (including four neighboring bps for spacing; the contribution of these additional bps was only tested for Rh4) from the broadly expressed ninaC, rdgA, or trpl drove broad expression in all photoreceptors, consistent with previous results. In sharp contrast, multimerized RCSI motifs drove expression in subsets of photoreceptors. The RCSI of Rh3 and Rh6 contains a K₅₀ motif, a binding site for K₅₀ homeodomain proteins such as the Dve repressor or the Otd activator. Expression of [Rh3 RCSI]₄ and [Rh6 RCSI]₄ was biased to inner photoreceptors: [Rh3 RCSI]₄ mediated restricted expression in R8 and R7, with a strong bias toward the pR7 subset, where Rh3 is normally expressed. This pattern is complementary to the expression of Dve, which is indeed responsible for the restricted expression as [Rh3 RCSI]₄ drove a broad, P3-like pattern in dve mutants. [Rh6 RCSI]₄ drove restricted expression in R8s and R7s; expression in R1 to R6 was very weak in comparison to P3 motifs, which was due to dve-dependent repression (Rister, 2015).

[Rh1 RCSI]₄ drove variable expression in R1 to R6, where Rh1 is expressed. This outer photoreceptor-specific pattern is complementary to the inner photoreceptor expression of [Rh3 RCSI]₄. Rh4 has the same RCSI as Rh1. However, adding the synergistic 3' RCSII motif led to expression in yR7s, where Rh4 is expressed. Although [Rh5 RCSI]₄ was not sufficient for reporter expression, adding three K₅₀ motifs to a single Rh5 RCSI ([K₅₀]₃ + [Rh5 RCSI]₁) led to expression in R8 and pR7 photoreceptors (Rister, 2015).

In summary, the RCSI motifs of specific Rhs differ from palindromic P3 motifs in broadly expressed genes: They drive expression that is biased toward the endogenous Rh expression patterns. It is shown below that full subtype specificity and activation often requires the repetition of motifs that are present in the RCSI (Rister, 2015).

As specific RCSI motifs directed restricted expression in different photoreceptor subsets, albeit with incomplete subtype specificity and with some variability in expression levels, it was asked whether the single-bp differences are required for subtype specificity in a wild-type promoter context and which other motifs are required for full restriction. The K₅₀ (Otd/Dve) motifs (TAATCC) were mutated to Q₅₀ (Pph13) motifs (TAATTG/A) to disrupt repression while preserving RCSI-mediated activation. Mutating the Rh3 RCSI resulted in an expansion to yR7s, where Dve is present at low levels. Mutating the Rh6 RCSI caused derepression in R1 to R6 and the ocelli. Rh3 and Rh6 have K₅₀/Dve repressor motifs repeated upstream, and mutation of individual motifs also caused derepression in yR7s and R1 to R6 and the ocelli, respectively. Taken together, single-bp changes create K₅₀ motifs in the Rh3 and Rh6 RCSI, which are required for subtype-specific expression together with their upstream repeats (Rister, 2015).

The importance of the disrupted P3 palindrome-i.e., the imperfect 3' homeodomain binding motif in the RCSIs of Rh1 toRh5 was examined. Creating a palindromic motif in theRh3 RCSI (TAATCCAATTC->TAATCCAATTA) caused derepression in yR7s that depended on Pph13. Therefore, derepression appears to be due to increased activation through the newly created Q₅₀/Pph13 site. The same ATTC→ATTA mutation in the Rh5 RCSI led to partial derepression in yR8s. This single-bp change created abinding site for the activator Otd (AGATTA), and indeed derepression in yR8s was lost in otd mutants, as was activation in pR8s (Rister, 2015).

The 3' ATTC motif in the RCSI of Rh3 and Rh5 is repeated upstream. Mutating the upstream repeat without creating a Q₅₀/Pph13 site (ATTC->CAAA) also caused derepression in yR7s (Rh3) or yR8s (Rh5). Mutating both ATTCs of Rh5 enhanced derepression into almost all yR8s. Therefore, this study has identified repressor motifs in the RCSIs of four Rhs (K₅₀/Dve motifs in Rh3/Rh6 and ATTC motifs in Rh3/Rh5). These motifs are repeated upstream within less than 100 bps and are required for full subtype specificity (Rister, 2015).

A single-bp ATTT->ATTA mutation in the Rh4 RCSI caused derepression in R1 to R6, pR7s, and the ocelli. The correct pattern was restored by crossing the mutant Rh4 reporter in a Pph13 mutant background, indicating that the A -> T change prevents Pph13 from overcoming repression in the 'wrong' photoreceptor subsets, as was the case for Rh3 and Rh6. The same mutation in the Rh1 RCSI caused no detectable derepression. Replacing two bps in the RCSI of the ocelli-specific Rh2 to obtain a Q₅₀/Pph13 site led to derepression in R1 to R6 photoreceptors that depended on Pph13 (Rister, 2015).

In vivo data revealed that a cell-fate decision requires single-bp differences in RCSI motifs. They complement previous findings in cell culture that subtle sequence differences in a glucocorticoid receptor or nuclear factor κB (NF-κB) binding site can specify the mode of transcriptional regulatio and that small differences in binding-site sequences can lead to distinct Hox specificities in vivo and in vitro. (1) Single bps in RCSI prevent binding of dimers of broadly expressed activators such as Pph13, tipping the balance of activator/repressor binding. This weakened activation allows repressors to prevent activation in other photoreceptor subtypes. (2) They generate specific combinations of overlapping activator and repressor motifs, often repeated upstream to provide robust expression and full subtype specificity. Creating overlap of activator and repressor motifs is an efficient way of blocking a key activator site in the 'wrong' cell types that express a repressor, especially because the RCSI motifs are very close to the transcription start site and repression there could block other activators. The precise tuning of RCSI motifs within their respective promoter context leads to incompatibility in other Rh promoters, as revealed by RCSI swap experiments: Replacing a given RCSI with another one resulted in two main outcomes: loss of expression or derepression in specific subsets of photoreceptors (Rister, 2015).

The RCSI/P3 motif resembles 'terminal selector' motifs that allow the coordinated expression of effector genes that define a particular neuron type. Yet, RCSI motifs exhibit additional layers of regulation that are integrated in a single regulatory element, as their sequence is modified for subtype specificity. Mutating a cis-regulatory motif in many cases appears to be the shortest evolutionary path toward a novel phenotype. Although it was found that it is possible in some cases to eliminate ectopic expression by removing the broadly expressed activator Pph13, this simultaneously causes a loss of expression of several broad phototransduction genes, defects in photoreceptor morphology, and a severe loss of light sensitivity (Rister, 2015).

It is proposed that the modification of a P3-type motif into different RCSI-type motifs allowed partitioning Rh expression to different subtypes of photoreceptors. This opened the possibility to discriminate wavelengths and likely conveyed a selective advantage. In this model, P3 motifs represent a positive regulatory element shared by ancestral genes that were expressed in all photoreceptors. This regulation is conserved, as the promoter of the long-wavelength Rh, as well as Gβ76C that are both expressed in all photoreceptors in the beetle Tribolium, contain a palindromic P3-type motif and depend on Pph13 (Rister, 2015).

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).

Cell cycle progression and differentiation are highly coordinated during the development of multicellular organisms. The mechanisms by which these processes are coordinated and how their coordination contributes to normal development are not fully understood. This study determined the developmental fate of a population of precursor cells in the developing Drosophila melanogaster retina that arrest in G2 phase of the cell cycle and investigated whether cell cycle phase-specific arrest influences the fate of these cells. Retinal precursor cells that arrest in G2 during larval development were shown to be selected as sensory organ precursors (SOPs) during pupal development and undergo two cell divisions to generate the four-cell interommatidial mechanosensory bristles. While G2 arrest is not required for bristle development, preventing G2 arrest results in incorrect bristle positioning in the adult eye. It is concluded that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. The results suggest that the control of cell cycle progression refines cell fate decisions and that the relationship between these two processes is not necessarily deterministic (Meserve, 2017).

This study developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. FlyBong was validated by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per), Clock (Clk), and cycle (cyc). The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization to cocaine in individual Drosophila. Because of its high-throughput nature, FlyBong can be used in genetic screens or in selection experiments aimed at the unbiased identification of functional genes involved in acute or chronic effects of volatilized psychoactive substances (Filosevic, 2018).

Smallish (Smash; CG43427), the Drosophila homologue of human LIM domain only 7 (LMO7), is a key regulator of Drosophila embryogenesis associated with planer cell polarity and actomyosin contractility at the zonula adherence. Although smash mRNA is expressed in several tissues during Drosophila development, only Smash function at the adherence junction in the embryonic epithelial cells has been reported. This study demonstrated that the knockdown of smash in eye imaginal discs induced morphological aberrations in adult compound eyes that were associated with increased apoptosis. Furthermore, immunohistochemical analyses revealed that Smash localized to the nucleus in several tissues, including eye imaginal discs. The knockdown of smash in eye imaginal discs down-regulated the expression of the otefin and bocksbeutel genes as well as the Drosophila homologue of the emerin gene, which is a target of LMO7. Collectively, these results indicate that Smash functions in proper Drosophila eye development mediated by the regulation of ote and bocks gene expression (Tanaka, 2018).

Robustness in development allows for the accumulation of genetically based variation in expression. However, this variation is usually examined in response to large perturbations, and examination of this variation has been limited to being spatial, or quantitative, but because of technical restrictions not both. This study bridged these gaps by investigating replicated quantitative spatial gene expression using rigorous statistical models, in different genotypes, sexes, and species (Drosophila melanogaster and D. simulans). Using this type of quantitative approach with molecular developmental data allows for comparison among conditions, such as different genetic backgrounds. This approach was applied to the morphogenetic furrow, a wave of differentiation that patterns the developing eye disc. Within the morphogenetic furrow, focus was placed on four genes, hairy, atonal, hedgehog, and Delta. Hybridization chain reaction quantitatively measures spatial gene expression, co-staining for all four genes simultaneously. Considerable variation was found in the spatial expression pattern of these genes in the eye between species, genotypes, and sexes. It was also found that there has been evolution of the regulatory relationship between these genes and that their spatial interrelationships have evolved between species. This variation has no phenotypic effect, and could be buffered by network thresholds or compensation from other genes. Both of these mechanisms could potentially be contributing to long term developmental systems drift (Ali, 2019).

DNA elements act across long genomic distances to regulate gene expression. During transvection in Drosophila, DNA elements on one allele of a gene act between chromosomes to regulate expression of the other allele. Little is known about the biological roles and developmental regulation of transvection. The stochastic expression of spineless (ss) in photoreceptors in the fly eye was investigated to understand transvection. It was determine a biological role for transvection in regulating expression of naturally occurring ss alleles. DNA elements were identified required for activating and repressing transvection. Different enhancers participate in transvection at different times during development to promote gene expression and specify cell fates. Bringing a silencer element on a heterologous chromosome into proximity with the ss locus "reconstitutes" the gene, leading to repression. These studies show that transvection regulates gene expression via distinct DNA elements at specific timepoints in development, with implications for genome organization and architecture (Urban, 2023).

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. This study identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. These factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate (Neal, 2020).

Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. This study investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes. These data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye (Gershman, 2020).

Little morphological information is available about subretinal pigment shields in insect compound eyes, especially ultrastructural information. The latter is however needed in order to detect possible smallest projections that emanate from pigment-granule-bearing cells and pass through the basal matrix (BM), but that are not visible in light micrographs. Previous work on the subretinal pigment shield in Drosophila melanogaster suggests that the pigment cell population located below the BM is closely associated with secondary and tertiary pigment cells. Whether these cells stay in connection throughout life with the subretinal regions via thin projections that pass through the fenestrae of the BM, or whether the subretinal parts later become separated during eye development remained so far unknown. This investigation of the periphery of the BM by three-dimensional reconstruction based on serial-sectioning transmission electron microscopy has revealed that the secondary and tertiary pigment cells possess thin projections that pass through the fenestrae of the BM and thus connect the cellular regions above and below the BM in the adult compound eye. The subretinal pigment shield of D. melanogaster is therefore of retinal origin and is not composed of additional subretinal pigment cells. The maintained bond allows the active displacement of pigment granules below the BM during the process of dark and light adaptation of the compound eye (Mohr, 2020).

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. This study used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. The CG lineage was found to segregate from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs' differentiation into WG. Interestingly, it was found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Thes findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons (Tsao, 2020). r

The size of organs is critical for their function and often a defining trait of a species. Still, how organs reach a species-specific size or how this size varies during evolution are problems not yet solved. This study investigated the conditions that ensure growth termination, variation of final size and the stability of the process for developmental systems that grow and differentiate simultaneously. Specifically, a theoretical model is presented for the development of the Drosophila eye, a system where a wave of differentiation sweeps across a growing primordium. This model, which describes the system in a simplified form, predicts universal relationships linking final eye size and developmental time to a single parameter which integrates genetically-controlled variables, the rates of cell proliferation and differentiation, with geometrical factors. The predictions of the theoretical model showed good agreement with previously published experimental results. A new computational model was developed that recapitulates the process more realistically and concordance was found between this model and theory as well, but only when the primordium is circular. However, when the primordium is elliptical both models show discrepancies. This difference is explained by the mechanical interactions between cells, an aspect that is not included in the theoretical model. Globally, this work defines the quantitative relationships between rates of growth and differentiation and organ primordium size that ensure growth termination (and, thereby, specify final eye size) and determine the duration of the process; identifies geometrical dependencies of both size and developmental time; and uncovers potential instabilities of the system which might constraint developmental strategies to evolve eyes of different size (Lobo-Cabrera, 2021).

Embryonic development leads to the reproducible and ordered appearance of complexity from egg to adult. The successive differentiation of different cell types that elaborate this complexity results from the activity of gene networks and was likened by Waddington to a flow through a landscape in which valleys represent alternative fates. Geometric methods allow the formal representation of such landscapes and codify the types of behaviors that result from systems of differential equations. Results from Smale and coworkers imply that systems encompassing gene network models can be represented as potential gradients with a Riemann metric, justifying the Waddington metaphor. This study extends the representation to include parameter dependence and enumerate all three-way cellular decisions realizable by tuning at most two parameters, which can be generalized to include spatial coordinates in a tissue. All diagrams of cell states vs. model parameters are thereby enumerated. The study unifies a number of standard models for spatial pattern formation by expressing them in potential form (i.e., as topographic elevation). Turing systems appear nonpotential, yet in suitable variables the dynamics are low dimensional and potential. A time-independent embedding recovers the original variables. Lateral inhibition is described by a saddle point with many unstable directions. A model for the patterning of the Drosophila eye appears as relaxation in a bistable potential. Geometric reasoning provides intuitive dynamic models for development that are well adapted to fit time-lapse data (Rand, 2021).

The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. This study shows that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function (Wang, 2022).

The Blimp-1 transcription factor has been shown to play important regulatory roles in the development of many cell types and organs in both flies and mammals. The only reported function for Blimp-1 in the mouse retina is to stabilize the photoreceptor cell fate; however, this study found that Drosophila Blimp-1 acts at a later stage of retinal development to promote the terminal differentiation of several cell types. It has no visible function in photoreceptors, but acts in the cone cells to enable normal photoreceptor rhabdomere extension and in the higher order pigment cells to promote the formation of a biconvex corneal lens. Blimp-1 controls the terminal differentiation of these cells by regulating a battery of genes that encode likely structural components of the eye as well as Slbo and other transcription factors. Upregulation of Blimp-1 expression in the retina requires ecdysone signaling, illustrating a role for steroid hormones in regulating sensory organ morphogenesis (Wang, 2022).

Although humans and Drosophila have shared common ancestors only in the very distant past and their lenses have distinct molecular compositions, both species evolved bi-convex lens shapes that enable them to focus light effectively onto the retina. The fly corneal lens cannot change its curvature through muscle contractions as the human lens does; to compensate for its fixed parameters, the fly retina is able to detect light throughout its depth. However, the thickness of the retina is insufficient to compensate for the massive reduction in the refractive power of the corneal lens resulting from the plano-convex shape caused by Blimp-1 loss-of-function, especially as loss of Blimp-1 also reduces retinal thickness. Loss of curvature on the external surface of Blimp-1 mutant corneal lenses is more detrimental to their refractive power than flattening of the internal surface facing the pseudocone would be, as the refractive indices of the corneal lens and pseudocone are similar to each other, but quite different from that of air (Wang, 2022).

Although major corneal lens proteins such as Crystallin and Retinin are produced by the cone and primary pigment cells, Blimp-1 acts in the more peripheral secondary and tertiary pigment cells to generate the external curvature of the corneal lens. Blimp-1 has been shown to regulate chitin deposition during embryonic cuticle formation and tracheal maturation, and chitin is a major component of the corneal lens that may determine its shape. Blimp-1 also controls the expression of numerous cuticle proteins that could be secreted from higher-order pigment cells to give the periphery of the corneal lens a composition distinct from the center. Besides regulating the expression of direct structural components of the corneal lens, Blimp-1 could contribute to the bi-convex shape by regulating the forces that pigment cells exert on the periphery of the corneal lens through its effects on the expression of cell surface and cytoskeletal proteins (Wang, 2022).

In addition to its effect on corneal lens shape, Blimp-1 loss-of-function causes a defect in photoreceptor morphology. Instead of extending throughout the depth of the retina, rhabdomeres are absent from the proximal retina, where the photoreceptors form stalk-like structures lacking rhabdomere markers. Photoreceptor nuclei are also displaced to the proximal ends of the rhabdomeres. During development, photoreceptors interact with cone cells as they undergo a 90 ° rotation of their apical membrane surface. Microvilli projecting from this apical membrane subsequently form rhabdomeres that extend proximally down the retina, growing to ~90 &mi;m in length. As cone cells are known to structurally and physiologically support photoreceptors during development, loss of Blimp-1 in these cells may affect their ability to contribute to photoreceptor development. Genes encoding cell surface, cytoskeletal and secreted proteins that this study found to be regulated by Blimp-1, including trol, could promote rhabdomere extension or attachment to an ECM produced by the cone cell feet (Wang, 2022).

Loss of Blimp-1 leads to a dramatic upregulation of the transcription factor Slbo in non-neuronal retinal cells. Overexpressing slbo in the eye results in a plano-convex lens and shortened rhabdomeres, similar to what is observed in Blimp-1 mutants. slbo overexpression could thus contribute to the Blimp-1 loss-of-function phenotypes. However, removing slbo is not sufficient to restore normal retinal development in the absence of Blimp-1, indicating that at least one crucial target gene requires direct input from Blimp-1 in addition to the absence of Slbo. Two distinct mechanisms, one regulated by Slbo and one by loss of Blimp-1 independently of Slbo, can thus lead to this constellation of phenotypes (Wang, 2022).

Most previous studies of slbo have focused on its requirement for normal border cell migration in the ovary and have shown that its overexpression is detrimental to most other tissues. As the eye is quite distinct from the ovary in its structure and morphogenesis, it is surprising that only a single transcription factor prevents slbo from being expressed there. It is more common to find a factor that is poised for expression when a single repressor is removed in tissues that share a common origin or developmental pathway. For example, knot is required for wing patterning but is repressed by Ultrabithorax in the haltere disc, which is a structure homologous to the wing disc. It is not known what activators drive slbo expression in the absence of repression by Blimp-1. In the ovary, slbo is activated by JAK/STAT signaling, which acts early in eye development to promote growth of the larval eye disc, but is not known to function in the pupal retina. The mouse slbo homologue Cebpa is activated by Blimp-1 in uterine tissues, suggesting possible conservation of the regulation of C/EBP genes by Blimp-1, but not of the direction of this effect (Wang, 2022).

Given the presence of Blimp-1 at the periphery of the developing mouse cornea, these results raise the intriguing possibility that its role in regulating the curvature of refractive surfaces is conserved. Further investigation of this hypothesis may provide insight into the genesis of the refractive disorders that afflict a growing number of people (Wang, 2022).

Bunker, J., Bashir, M., Bailey, S., Boodram, P., Perry, A., Delaney, R., Tsachaki, M., Sprecher, S. G., Nelson, E., Call, G. B. and Rister, J. (2023). Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway. Front Cell Dev Biol 11: 1058961. PubMed ID: 36960411

During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORbeta activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. This study shows that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORbeta promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, this study identified a novel interaction between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision (Bunker, 2023).

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wg^RNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development (Raj, 2020).

Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing. Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals, and can function to repress transcription. The genome-wide studies have revealed that  ~10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters. In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways, it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila. However, the biological relevance of transcriptional pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood (Raj, 2020).

M1BP regulates retinal determination and MF progression in developing eye This study tested fthe role of transcription pausing factor, M1BP during Drosophila eye development. Downregulation of M1BP levels in the developing eye was found to result in strong suppression of eye fate, however, gain-of-function of M1BP did not affect the eye fate suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, no domain constraint in eye suppression function was seen when M1BP levels were downregulated. In addition, when M1BP levels were downregulated (ey > M1BP^RNAi) the expression of retinal determination and differentiation genes were strongly downregulated. Interestingly, it was found that protein encoded by RD genes were downregulated in ey > M1BP^RNAi background. Therefore, M1BP may not be affecting RD gene expression directly (Raj, 2020).

During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it. The two signals dpp and hh plays an important role in initiation and progression of MF. Downregulation of M1BP affects retinal differentiation as well as progression of MF. It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. This study screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc (Raj, 2020).

The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, targets were sought of M1BP transcriptional pausing function using the candidate gene approach. It was found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated. This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression. Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change when cells are treated with M1BP^RNAi (Raj, 2020).

To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BP^RNAi eye imaginal disc, this study also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGGTCACACTR) has been reported earlier. This sequence was used for MEME analysis to screen for M1BP binding sites were found in the wg gene and regulatory region (Raj, 2020).

Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development. During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc. Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development. This study found that in ey > M1BP^RNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate. Further, downregulation of wg levels, using wg^RNAi, in ey > M1BP^RNAi background rescued the eye suppression phenotype. This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Raj, 2020).

Higher levels of Wg are known to trigger developmental cell death in the developing eye field. Interestingly, in ey > M1BP^RNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene in ey > M1BP^RNAi background showed rescue of eye suppression phenotype. However, these P35 mediated rescues of ey > M1BP^RNAi were not as significant as seen with wg^RNAi. This suggests that Wg might be regulating eye fate through hth induction and eye field size by triggering caspase mediated cell death. In order to rule out that these in ey > M1BP^RNAi phenotypes are not affected by reduced cell proliferation rates, levels were tested of pH3 in these developing eye fields. It was found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye (Raj, 2020).

These results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development. However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand whether M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, the downregulation of M1BP function was studied in bi-Gal4 domains of wing imaginal disc. Whether this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc was tested. A bi-GAL4 driver which drives the expression of a transgene in wing imaginal disc was used. Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BP^RNAi) exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc. Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling. In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila (Raj, 2020).

A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5^W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis. Interestingly, the mutant allele of Spt5 (foggy^m806) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation. Therefore, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development (Raj, 2020).

Tiling patterns are observed in many biological structures. The compound eye is an interesting example of tiling and is often constructed by hexagonal arrays of ommatidia, the optical unit of the compound eye. Hexagonal tiling may be common due to mechanical restrictions such as structural robustness, minimal boundary length, and space-filling efficiency. However, some insects exhibit tetragonal facets. Some aquatic crustaceans, such as shrimp and lobsters, have evolved with tetragonal facets. Mantis shrimp is an insightful example as its compound eye has a tetragonal midband region sandwiched between hexagonal hemispheres. This casts doubt on the naive explanation that hexagonal tiles recur in nature because of their mechanical stability. Similarly, tetragonal tiling patterns are also observed in some Drosophila small-eye mutants, whereas the wild-type eyes are hexagonal, suggesting that the ommatidial tiling is not simply explained by such mechanical restrictions. If so, how are the hexagonal and tetragonal patterns controlled during development? This study demonstrates that geometrical tessellation determines the ommatidial tiling patterns. In small-eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. It is proposed that the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes (Hayashi, 2022).

Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. In this study RNAi-based knockdowns were performed of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, two optically different compound eyes were tested: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, multiple aspects of ocular formation were disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, these findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role (Rathore, 2023).

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. This study found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets (Bhattacharya, 2017).

Differences were observed between how Notch signaling regulates the SMW compared to how Notch regulates photoreceptor differentiation (see Model of the cell cycle and cell fate specification in the morphogenetic furrow). The Notch pathway suppresses the specification of photoreceptor cells in a manner similar to regulation of neurogenesis by Notch in many other tissues. That is, activation of the transmembrane ligand Delta by the ubiquitin ligase Neuralized leads allows Delta to activate Notch, leading to release of the Notch intracellular domain, which then acts in a nuclear complex with Su(H) and Mastermind to induce the transcription of the E(spl)-C family of transcriptional repressors and prevent neural fate specification and differentiation. By contrast to the pathway regulating neural differentiation, cell cycle entry in the SMW occurred in the absence of the E(spl)-C. This suggested that a distinct transcriptional target of Su(H) is involved, but unusually, this Su(H) function did not seem to require the co-activator Mam. Although most studies find that Mam is required for transcriptional activation by Su(H), mutations in mam often have weaker phenotypes than other neurogenic genes. It is possible that Mam protein might exhibit exceptionally strong perdurance, but there is also in vitro evidence for mam-independent transcriptional activation by Su(H) (Bhattacharya, 2017).

SMW entry also occurred in the absence of neur. In the case of neurogenesis, cells lacking neur show a weaker neurogenic phenotype than cells lacking other components of the pathway, both in the embryonic CNS and in the eye, which is thought to reflect the activities of two other ubiquitin ligases, Mindbomb and Mindbomb2. No effect was found on the SMW of mutated mib1, but neither mib2 nor cells lacking two or more of these possibly redundant ubiquitin ligases were examined (Bhattacharya, 2017).

Eoles of ligands were examined in more detail. It has been reported that SMW entry depended on Delta, which is also the ligand necessary for regulation of neurogenesis in the eye as in other parts of the nervous system, but although their figures show an obvious reduction in cell cycle entry in the clones of cells lacking Dl, some cell cycle entry still seems to occur. Those experiments were repeated using cells lacking both Dl and the other known ligand Ser, with similar results, ie, cell cycle entry was obviously disrupted in clones lacking both Notch ligands, nevertheless some cells still entered the cell cycle. A potential complicating factor is that reducing Notch function typically results in excessive neurogenesis, raising the question of whether some Dl Ser mutant cells were prevented from entering the cell cycle by differentiation as photoreceptors. The excess neurogenesis of such clones was partially suppressed by expression of Argos or Ras-DN. Although there is clearly a considerable disruption of the SMW in the absence of N ligands, there is also clear evidence of cell cycle entry, occurring at approximately the same time as the normal SMW (Bhattacharya, 2017).

There are at least three possible explanations for these findings. First, it possible that another ligand besides Dl or Ser activates N in the SMW. Secondly, it has been reported that Dl can signal across several cell diameters using filopodia. During neurogenesis, rescue of Dl mutant clones appears to extend beyond cells immediately adjacent to wild type cells. Signaling across many cell diameters would be required to account for the cell cycle progression seen in Dl Ser mutant clones. Thirdly, Notch can be activated independently of ligands if delivered to certain cellular compartments. Recently it has been suggested that such ligand-independent activation is suppressed by cis-inactivation, in which ligands inhibit N signaling in the same cells, so that ligand-independent activation can be revealed in cells lacking Dl and Ser. Cis-inactivation is potentially an important contributor to the patterning of Notch-mediated lateral inhibition during neurogenesis, but direct evidence of such a role has not yet been obtained. If ligand-independent signaling is occurring in Dl Ser clones, some other pathway must be active near the SMW to explain why ligand-independent signaling cell cycle entry was limited to cells at the same stage posterior to the furrow as those that undergo the normal, ligand-dependent SMW (Bhattacharya, 2017).

The cell cycle targets of Notch are of some interest. Many Drosophila cell cycles depend on regulated transcription of Cyclin E. Cyclin E is clearly required for the SMW suggested that Cyclin E might not be limiting for SMW cell cycle entry, however, because Cyclin E was seen to accumulate more in the differentiating photoreceptor precursors that do not enter the SMW. Alternatively, the lack of Cyclin E accumulation in SMW cells might reflect Cyclin E protein instability in S phase, with accumulation only in differentiating cells that can't enter the cell cycle. A Cyclin E transcriptional reporter appears to accumulate in the same cells as the Cyclin E protein, however, which is not indicative of post-translational regulation (Bhattacharya, 2017).

It has been suggested that Cyclin A was part of the Notch dependent machinery promoting cell cycle entry in the SMW, although it was concluded that it could not be the only important Notch target. What was observed, however, was dependence of Cyclin A protein levels on Notch signaling that exactly paralleled the dependence of S-phase entry on Notch signaling in various genotypes. Like Cyclin B, Cyclin A protein is degraded by APC/Cyclosome activity during G1, so that Cyclin A protein cannot accumulate until the G1/S transition. Therefore these results are also expected if Cyclin A simply accumulates whenever the G1/S transition has occurred, and is not a direct target of Notch signaling (Bhattacharya, 2017).

In mammalian cells, c-Myc appears to be an important Notch target. Indeed, human Notch was first identified as a proto-oncogene that causes leukemia through c-Myc. c-Myc is an activator of the nucleolar protein fibrillarin, which transiently increases during the SMW. Although Dmyc is important for growth in Drosophila, so that clones of myc null mutant cells in the eye disc are small, they nevertheless enter the SMW at the normal time, indicating that myc is unlikely to be the critical Notch target in the SMW (Bhattacharya, 2017).

Emc is a transcriptional target of N signaling in the eye as in other tissues. Even though Emc protein levels seem to be less affected by N signaling than is emc RNA, it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry. Contrary to this model, previous studies have reported that Da is in fact required for the SMW. We investigated this potential role for emc directly, finding no evidence that emc was required for the SMW. These studies confirmed previous findings that da is required for S phase entry into the SMW, although not apparently for cell cycle activity anterior to the morphogenetic furrow (Bhattacharya, 2017).

The cell-autonomy of da requirement in the cell cycle had not been determined previously. da mutant cells sometimes enter the SMW at the posterior boundaries of da clones, where they were adjacent to wild type cells. This suggested that da was required for the production of a short-range non-autonomous signal that is required for SMW entry, and could be provided to da mutant cells that were near to wild type cells (Bhattacharya, 2017).

It is instructive to compare the effect of da on the cell cycle to that of ato. Ato is the heterodimer partner of Da that is required for R8 specification. Da is also required independently of ato to specify R2,R3,R4 and R5. Interestingly, this requirement in R2-5 seems to be incomplete, since some da mutant R2-5 cells do differentiate at reduced frequencies. Because of this requirement for da outside R8, R1-7 photoreceptor cells can differentiate near borders of ato mutant clones, but fewer photoreceptors are expected near the borders of da mutant clones, and mostly of the R1, R6 and R7 cell types. Proneural genes often promote Notch ligand expression, and accordingly ato is required for normal Dl expression during these stages of eye development. Reduced Dl expression is a plausible explanation for the absence of the SMW within ato clones, and the more extensive requirement for da in photoreceptor differentiation and Dl expression may explain why non-autonomous rescue of the SMW is more pronounced near the borders of ato clones than near the borders of da clones (Bhattacharya, 2017).

A model is present for SMW. Unpatterned proliferation of the eye imaginal disc is terminated ahead of the morphogenetic furrow by Hh and Dpp signaling. Hh and Dpp affect many aspects of eye development including expression of master regulators of retinal determination such as Eyeless, Teashirt, Dachshund, and Sine Oculis, as well as Homothorax. Cells posterior to the furrow can still respond to growth, because extra cell cycles are driven in unspecified cells by overexpression of CycD/cdk4, Inr, or myc, as well as by Cyclin E. It is possible that Notch regulates cellular growth in the SMW, since there appears to be a small but discernible increase in nucleolar size at this stage, and progenitor cells are not noticeably smaller after dividing, indicating that growth accompanies the SMW cell division. The SMW, however, does not depend on myc. It appears to depend on a transcriptional target of Su(H) outside of the E(spl)-C of bHLH genes and that can be transcribed in the absence of Mam. The possibility of a function of Su(H)/Nicd other than transcription cannot be ruled out. The SMW clearly depends on CycE, but CycE may not be the N-dependent gene required for SMW entry, because it is not obviously elevated in SMW cells. Contrary to a recent proposal, the SMW does not depend on the HLH gene emc and is not inhibited by the bHLH protein Da. Instead da is required for entry into the SMW. The positive requirement for da is cell-nonautonomous, as is the requirement for its heterodimer partner ato, and consistent with the role of ato and da in promoting Dl expression during retinal differentiation, which is likely to account for the cell cycle defect. Surprisingly, the requirement for Dl in the SMW cell cycle was not absolute, either because an unidentified ligand exists, long-range cell-nonautonomy occurs, or Dl (and Ser) may contribute to cis-inactivation of N, so that some ligand-independent Notch signaling could occur in the unphysiological circumstance that Notch ligands are completely absent (Bhattacharya, 2017).

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that normal Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of mir-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. mir-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently de-represses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, this study reveals a rare setting of developmental cell specification that substantially requires miRNA control (Duan, 2018).

Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). This study using a novel temperature-sensitive (ts) allele, showed that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1^ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1^ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1^ts mutants. However, Sac1^ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo. Together, these data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner (Del Bel, 2018).

Undifferentiated epithelial cells are patterned and specified during development to yield highly ordered tissues composed of multiple cell types. Patterning and differentiation within an epithelium are driven in part by cell surface adhesion molecules that promote intercellular interactions. Defects in cell-cell adhesion lead to developmental abnormalities and contribute to disease progression. For example, the mammalian Irre cell recognition module (IRM) adhesion proteins NEPH1 (KIRREL) and nephrin (NPHS1) are required in the kidney for development and maintenance of the filtration barrier or slit diaphragm. Despite their importance in animal development and physiology, little is known about how trafficking and delivery of cell surface adhesion molecules such as IRM proteins is achieved (Del Bel, 2018).

The adult Drosophila eye contains ~750 individual units called ommatidia. Prior to pupariation, each ommatidium consists of eight photoreceptor (PR) cells and four cone cells, surrounded by undifferentiated interommatidial cells (IOCs). During the first half of pupal eye development, 0-42 h after puparium formation (APF), IOCs undergo dynamic morphogenetic changes to give rise to two primary pigment cells (1°pc), six secondary pigment cells (2°pc), three tertiary pigment cells (3°pc) and three bristles, arranged in a honeycomb lattice. These cells support and optically isolate individual ommatidia (Del Bel, 2018).

Specification and organization of Drosophila retinal cells requires IRM protein function. 1°pc express the nephrin homologs Hibris (Hbs) and Sticks and stones (Sns), whereas IOCs express the NEPH1 homologs Roughest (Rst) and Kin of Irre (Kirre, also called Dumbfounded). Between 24 and 30 h APF, all four proteins localize to the plasma membrane (PM) of these cells at the 1°pc:IOC border, and heterophilic binding of Rst and Kirre with Hbs and Sns is needed for specification and morphogenesis of 2°pc and 3°pc (2°/3°pc). Mutations that affect cell surface accumulation of Rst result in rough eyes and reduced levels of pigmentation as a result of defects in 2°/3°pc differentiation (Del Bel, 2018).

Phosphoinositides, or phosphatidylinositol (PI) phosphates (PIPs), regulate essential cellular processes such as membrane trafficking and actin cytoskeletal organization. In the canonical PIP pathway, PI is phosphorylated by PI 4-kinases (PI4Ks) to generate PI 4-phosphate (PI4P), which is a precursor for other PIPs, including PI 4,5-bisphosphate [PI(4,5)P2], and serves as a potent signaling molecule, for example by recruiting effectors to the Golgi body to promote membrane trafficking. Downregulation of PI4P in specific membrane compartments also drives cellular events. For example, in budding yeast (Saccharomyces cerevisiae), PI4P dephosphorylation is required for targeted delivery of cargo to the PM by the exocyst complex. Drosophila has three PI4Ks, including a single type II enzyme (PI4KII) that generates PI4P at the trans-Golgi network (TGN) and on endosomes. PI4P levels are kept in check by the phosphoinositide phosphatase Sac1, a conserved transmembrane protein present in endoplasmic reticulum and Golgi membranes (Del Bel, 2018).

Previous work has shown that Sac1 is required for normal eye development in Drosophila (Wei, 2003b). Sac1 is essential for viability in Drosophila (Wei, 2003a,b). Flies transheterozygous for a hypomorphic allele and a null allele exhibit rough eyes with necrotic patches and drown in the food soon after eclosing (Wei, 2003b). Using the hypomorphic allele, which is shown to be temperature sensitive (ts), it was demonstrated that Sac1 plays a crucial role in patterning the retinal epithelium. IOCs of Sac1^ts flies cultured at 25°C exhibit a dramatic increase in PI4P levels and decreased levels of PM PI(4,5)P2. Although Rst is present at the cell surface of these IOCs, fixation on ice ex vivo results in re-distribution of Rst to enlarged structures containing the exocyst complex subunit Sec8, suggesting a microtubule defect. Indeed, Sac1^ts IOCs contain sparse, disorganized microtubules (MTs) that disappear when fixed on ice. These results thus identify a novel link between Sac1, PIP levels and microtubule stability in the developing Drosophila eye. Because Sac1 is conserved, these findings suggest a possible role for MT regulation by Sac1 in human development and disease (Del Bel, 2018).

Conditional alleles are powerful tools for studying the functions of essential genes. Using a temperature-sensitive allele of Sac1, this study shows that proper phospholipid regulation by Sac1 is required for normal development of the Drosophila retinal epithelium (Del Bel, 2018).

The results indicate that retinal patterning is highly sensitive to elevated levels of PI4P. Loss of Sac1 leads to increased Golgi PI4P and IOC patterning defects that are rescued by expression of catalytically active Sac1. Deletion of PI4KII suppresses Sac1^ts, whereas expression of a WT PI4KII transgene exacerbates the observed defects. Thus, Sac1 and PI4KII regulate a pool of PI4P that is required for retinal patterning. Loss of PI4KII itself results in mild patterning defects (i.e. occasional extra bristles and fused ommatidia), indicating that reducing PI4P levels also has consequences for retinal development. Although these data point to a clear role for PI4KII in signaling required for 2°/3°pc adhesion and patterning, the experiments do not rule out possible contributions of other PI4Ks to this process. Moreover, it is anticipated that, in addition to the Golgi, other pools of PI4P at the PM and elsewhere may be regulated by Sac1 (Del Bel, 2018).

Because PI4P is phosphorylated by PIP 5-kinases to produce PI(4,5)P2, Sac1^ts mutants might be expected to show increased levels of PI(4,5)P2 as well as PI4P. Strikingly, however, this study found that Sac1^ts mutants exhibit reduced levels of the PI(4,5)P2 marker PLCΔ-PH at the PM. Although this result is counterintuitive, similar observations have been reported in budding yeast, and the same same phenomenon in Drosophila is observed embryos homozygous for a lethal allele of Sac1. This observation is in contrast to other Drosophila tissues, where Sac1 loss was reported to have no effect on PI(4,5)P2 levels (Forrest, 2013; Yavari, 2010). It is possible that PIP 5-kinases lack access to the increased PI4P in Sac1 mutants, thereby preventing the expected increase in PI(4,5)P2, or perhaps increased PI4P triggers a feedback mechanism that activates PIP phosphatases or phospholipases, leading to PI(4,5)P2 breakdown. Thus, in addition to elevated PI4P levels, reduced levels of PI(4,5)P2 might contribute to the IOC patterning defects in Sac1^ts. Indeed, in preliminary results a genetic interaction was observed between Sac1 and the PI4P 5-kinase skittles, which could stem from reduced PI(4,5)P2 or increased PI4P levels, or a combination of the two (Del Bel, 2018).

Drosophila Sac1^ts mutants exhibit rough eyes, ommatidial mispatterning and abnormal 2°/3°pc morphology, phenotypes that resemble mutants for the IRM protein Rst. Maintenance of Rst at the 1°pc:IOC border is sensitive to ice fixation in Sac1^ts mutant retinas. Because MTs are labile under cold conditions, this suggested a MT defect in Sac1^ts. MTs are disorganized in Sac1^ts mutant IOCs fixed at RT, and even more dramatically affected in mutant IOCs fixed on ice. Moreover, Sac1^ts IOCs fixed on ice display enlarged Sec8-positive vesicles containing Rst, suggesting that Rst travels to the cell surface via an exocyst- and MT-associated pathway that is sensitive to cold fixation in Sac1^ts (Del Bel, 2018).

The simplest model to explain these data is that Rst is constantly turned over at the PM and requires continuous replenishment from internal stores, as has been reported for mammalian IRM proteins. This turnover appears to occur in both WT and Sac1^ts, as this study observed similar uptake of anti-Rst antibody in each. It remains possible that subtle defects in the activity or turnover of Rst (or other proteins) at the PM contribute to the observed defects in Sac1^ts. Indeed, compromised expression of the differentiation marker BA12-lacZ was observed in Sac1^ts IOCs. Defects in BA12-lacZ expression, as well as IOC number and specification, also occur in RstD mutants, which show a delay in Rst accumulation at the 1°pc:IOC border. Relevant to this point, it is noted that when quantification of Rst distribution after RT or cold fixation was independently validated at a later date, a slight but significant reduction was observed in Rst accumulation in Sac1^ts following RT fixation. However, as this difference was not observed in earlier experiments, it is not possible to conclude that there is a reproducibly significant decrease in Rst accumulation in Sac1^ts (Del Bel, 2018).

Sac1 was originally identified as a suppressor of yeast actin mutants. Thus, much of the literature has focused on how Sac1 loss affects the actin cytoskeleton. This observed that retinal cells in Drosophila Sac1^ts mutants exhibit normal actin organization during early stages of pupal eye development (24-30 h APF), yet show defects in MT stability and organization. Interestingly, both increased levels of PI4P and reduced levels of PI(4,5)P2 have previously been associated with defects in MT organization. The molecular mechanisms involved remain unknown, and further investigation will be required to determine the link between Sac1 and MT stability. However, the current observations suggest that regulation of the MT cytoskeleton by Sac1 may be more important when moving from fungal to animal models (Del Bel, 2018).

In summary, this study has characterized a requirement for Sac1 in maintaining PI4P and PI(4,5)P2 levels and promoting normal development of the Drosophila retina. Additionally, it was demonstrated that Rst distribution in Sac1^ts is sensitive to cold fixation, and that Sac1^ts exhibits a microtubule defect at 24 h APF. The results indicate that Rst is delivered to the PM via exocyst- and microtubule-based trafficking (which is cold sensitive in Sac1^ts) and is turned over at the PM, similar to its mammalian homologs. Further investigation will be required to unravel precisely how Sac1 regulates microtubules, and how this contributes to normal retinal development (Del Bel, 2018).

The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1^ts), this study shows that Sac1 is required for structural integrity of the Drosophila retinal floor. The βps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1(ts) retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1(ts) retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, these data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development (Griffiths, 2020).

Although they comprise a minor fraction of total cellular phospholipid content, phosphoinositides, also known as phosphatidylinositol phosphates (PIPs), act as essential coordinators of membrane function and identity (Balla, 2013). PIPs are derived from the precursor phosphatidylinositol, whose inositol head group can be phosphorylated at any of three positions to yield seven unique PIP species that recruit distinct sets of effector proteins. Through the localized activity of PIP kinases and phosphatases, these species are interconverted to maintain enrichment in different membranes and to regulate numerous PIP effector-driven processes (Balla, 2013; Griffiths, 2020).

Sac1 is a conserved phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates multiple stages in secretory trafficking, participates in cellular signaling pathways, and acts as the precursor for PI(4,5)P2 at the plasma membrane (PM). PI4P is produced in the PM and Golgi, respectively, by two conserved type III PI 4-kinases (PI4Ks), PI4KIIIα, and PI4KIIIβ. In addition, a type II PI4K (PI4KIIα) produces PI4P in the trans-Golgi network (TGN) and on endosomes, where it is important for endosomal trafficking (Griffiths, 2020).

In contrast to the distribution of PI4Ks and PI4P, Sac1 localizes primarily to the ER, as well as the cis-Golgi under growth-limiting conditions. Although seemingly capable of acting in trans on PI4P in neighboring membranes in some scenarios, Sac1 appears to predominantly depend on delivery of PI4P to the ER via nonvesicular lipid transport at membrane contact sites (MCS). For instance, oxysterol-binding protein (OSBP), which localizes to ER-trans-Golgi MCS through interactions with the ER-resident vesicle-associated membrane protein-associated protein VAP as well as PI4P in the trans-Golgi, delivers PI4P from the trans-Golgi to the ER in exchange for sterols. Hydrolysis of incoming PI4P by Sac1 maintains a low concentration of PI4P in the ER that is necessary for sustained PI4P/sterol countertransport in vitro, although this relationship appears more nuanced in vivo. OSBP-related proteins (ORPs), which are encoded by 11 genes in humans and three in flies, function similarly to OSBP but differ in their localization and lipid-binding preferences. Despite its essential function, how Sac1 regulates different aspects of cellular homeostasis during animal development is not fully understood (Griffiths, 2020).

In Drosophila, null Sac1 mutants exhibit embryonic lethality due to defects in cell shape and ectopically activated JNK signaling that prevent dorsal closure. JNK signaling defects are also observed in Sac1 clones in larval imaginal discs. Moreover, Sac1 regulates Hedgehog signaling by inhibiting recruitment and activation of Smoothened at the PM in a PI4P-dependent manner (Yavari, 2010; Jiang, 2016). Sac1 is also required for axonal pathfinding in the embryonic central nervous system, as well as for axonal transport and synaptogenesis in larval neurons (Lee, 2011; Forrest, 2013; Griffiths, 2020).

In addition, loss of Sac1 causes severe tissue disorganization and degeneration during eye development. The Drosophila eye is composed of ~750 unit eyes called ommatidia. Presumptive ommatidia arise early in pupal development, where they initially comprise clusters of medial/basal photoreceptors and apical cone cells surrounded by a disordered pool of undifferentiated interommatidial cells (IOCs). During the first half of the ∼96-h pupal stage, two IOCs per ommatidium differentiate into primary pigment cells (1°pc), which encircle the cone cells. The remaining IOCs subsequently differentiate into a lattice of secondary and tertiary pc (2°/3°pc) and sensory bristles that separate neighboring ommatidia or are removed by apoptosis by 42 h after puparium formation (APF). Changes in IOC shape and position during this stage require the Irre cell recognition module (IRM) adhesion proteins Roughest (Rst) and Hibris (Hbs), as well as their paralogues Kirre and Sticks and stones (Sns). Rst/Kirre and Hbs/Sns are orthologues of mammalian Neph1 and nephrin, which are needed for formation of the renal slit diaphragm as well as during myoblast fusion. After IOC patterning, during late stages of pupal eye development (42-96 h APF), the retina elongates fivefold, and laminated corneal lenses with underlying gelatinous pseudocones are secreted, giving the eye its characteristic adult appearance (Griffiths, 2020).

Previously work has examined the role of Sac1 in the developing Drosophila eye using a hypomorphic Sac1 allele that is temperature sensitive (Sac1^ts). Sac1^ts flies develop morphologically normal eyes when reared at 18°C, but display a rough eye phenotype caused by defective IOC sorting when reared at or above 23.5°C. This study shows that Sac1^ts eyes exhibit structural defects at the retinal floor and mislocalization of the βps-integrin Myospheroid (Mys), which is required for retinal floor adhesion. This defect is not due to a loss of cell polarity, as apical adherens junctions are unaffected. However, a novel secondary defect was identified in the distribution of Rst and Kirre, which are apical transmembrane proteins. At 42 h APF, Sac1^ts 2°/3°pc contain an excess of intracellular Rst and Kirre due to impaired endo-lysosomal trafficking and degradation. Sac1^ts 2°/3°pc also accumulate PI4P and F-actin on enlarged, basal endosomes and exhibit ER stress. Thus, this study has identified novel roles for Sac1 in regulating cellular homeostasis during tissue morphogenesis (Griffiths, 2020).

The Drosophila pupal eye represents a powerful system to examine protein trafficking and turnover. Patterning of retinal cells requires spatially and temporally regulated expression as well as correct subcellular distribution of cell surface proteins that mediate cell-cell contacts and determine tissue architecture. Dysregulation of these processes can produce structural defects, which frequently persist in the adult eye. This study has taken advantage of these circumstances to demonstrate the importance of Sac1 in basal delivery of the βps-integrin Mys, which is required for retinal floor integrity, as well as endo-lysosomal regulation and turnover of the apical patterning determinants Rst and Kirre. The results also highlight the importance of Drosophila Sac1 in ER homeostasis, as had been reported in yeast. This could be due to deregulation of PI4P, phosphatidylserine, and sterol levels, which would be expected to disrupt ER membrane charge and lipid order (Griffiths, 2020).

Given the similarities between mys mutants and Sac1^ts, loss of Mys at the basal grommets in Sac1^ts likely causes the retinal floor defects observed in the adult eye. In addition, this phenotype resembles the basal retinal degeneration observed in an ALS-associated vap mutant, suggesting the underlying cause could be similar. However, it is unclear why basal distribution of Mys is perturbed while apical polarity is not. In the Drosophila follicular epithelium, Rab10 activity has been shown to be important for the distribution of basement membrane proteins independent of overall apical-basal polarity, in a manner dependent on PI(4,5)P2 at the apical PM. Previously study observed a decrease in apical PI(4,5)P2 abundance in Sac1^ts retinas at 24 h APF (Del Bel, 2018), which it is speculated could perturb basal trafficking. Alternatively, aberrant distribution of basal F-actin in Sac1^ts could inhibit localization of Mys to the grommets. Why some transmembrane proteins are sensitive to reduced Sac1 activity while others are not remains an open question. It is also unclear whether Mys mislocalization is linked to endosome dysfunction in Sac1^ts (Griffiths, 2020).

Whereas PI3P and PI(3,5)P2 are the canonical phosphoinositide regulators of endosomal progression (Wallroth, 2018), PI4P production has also emerged as an important factor in cargo delivery to lysosomes. In mammalian cells, PI4P is generated on late endosomes by type II PI4Ks (Baba, 2019). PI4KIIα is important for Golgi-to-lysosome trafficking of LIMP-2, as well as PM-to-lysosome trafficking of LAMP-1, and these proteins accumulate in enlarged endosomes when PI4KIIα levels are reduced. Furthermore, in macrophages, PI4KIIα-mediated PI4P enrichment on phagosomes occurs concurrently with Rab7 recruitment and is necessary for phagosome acidification and subsequent fusion with lysosomes (Griffiths, 2020).

This study has shown that Sac1-dependent depletion of PI4P is also important for endosomal trafficking and degradation of transmembrane proteins from the PM. This is consistent with a recent report by Mao and colleagues (Mao, 2019), who found that in multiple larval Drosophila tissues, loss of VAP, which recruits OSBP and a subset of ORPs to MCS, increases endosomal PI4P levels and inhibits autophagic degradation. Null vap mutants exhibit decreased lysosomal acidification, as well as an increase in the abundance of lysosomes, endosomes, autolysosomes, autophagosomes, and Ref(2)P (Mao, 2019). The authors propose that increased PI4P abundance up-regulates endosome formation and progression, which causes lysosomes to become oversaturated with incoming cargo. Indeed, loss of Ubiquilin, which contributes to lysosome acidification, also delays autophagy and causes Ref(2)P buildup. Notably, this study observed increased Ref(2)P abundance in Sac1^ts retinas, which suggests a similar delay in autophagy. It is a compelling notion that increased PI4P levels in Sac1^ts could promote excessive fusion of endosomes with lysosomes, which would replicate the effect described by Mao (2019). However, the accumulation of Rst and Kirre in Sac1^ts, which do not appear to be concentrated in lysosomes based on the lack of colocalization between Rst and Arl8, could also be caused by impaired endosomal progression or maturation, though this might stem from downstream lysosomal dysfunction. Indeed, the enlarged endosomes observed in Sac1^ts lacked both Vps16a and Arl8, suggesting they were not caused by excessive fusion with lysosomes. Further analysis of PI4P in endosomal dynamics and maturation is warranted to determine the precise role of Sac1 in late stages of protein degradation (Griffiths, 2020).

This study also found that reduced Sac1 function leads to basal accumulation of F-actin-positive enlarged endosomes. In mammalian cells, loss of both VAP isoforms has been shown to induce F-actin comet formation on endosomes via PI4P-dependent recruitment of the WASH-ARP2/3 complex. Notably, these do not resemble the more uniform F-actin coating on Sac1^ts endosomes. Rather, the structures that were observed appear more reminiscent of a phenomenon termed actin-flashing, wherein phagosomes become coated in F-actin by WASP-ARP2/3 to delay fusion with lysosomes. Endosomal phenotypes similar to those in Sac1^ts have also been observed when Arf6 activity is perturbed; increased Arf6 activity activates PIP5K, which has been shown to produce PI(4,5)P2 on endosomes and lead to F-actin polymerization via WASP, whereas loss of Arf6 increases endosomal PI4P levels and perturbs endosomal recycling. Intriguingly, in Caenorhabditis elegans, Sac1 inhibits Arf6 by sequestering the Arf6-GEF Bris-1. However, it is unknown whether this interaction is conserved or, more broadly, how Sac1 influences F-actin polymerization on endosomes (Griffiths, 2020).

It is noteworthy that enlarged endosomes were restricted to basal regions in Sac1^ts. Positioning of endosomes and lysosomes is mediated by bidirectional transport along microtubules, which influences their acidity and function. In mammalian cells, Rab7 recruits RILP, which activates endosomal dynein motors to promote minus end-directed transport toward perinuclear microtubule organizing centers. PI4P is also required for RILP recruitment, which implies that excess PI4P could lead to perinuclear endosome accumulation. Although the single Drosophila RILP orthologue has been shown to bind Arl8 rather than Rab7, it is possible that PI4P influences late endosome transport through analogous Rab7 effectors. Additionally, previous work has shown that Sac1^ts 2°/3°pc precursors contain unstable microtubules at 24 h APF (Del Bel, 2018), which could affect microtubule-based endosome positioning later in development (although this study was unable to detect microtubule defects by immunostaining at 42 h APF). However, it is also possible that enlarged endosomes accumulate basally for other reasons or are simply excluded from narrower apical-medial regions on the basis of size. It remains to be discerned whether Rst accumulation and the appearance of enlarged endosomes, which co-occurred between 24 and 42 h APF, share a causal basis or represent distinct, parallel phenotypes of reduced Sac1 activity (Griffiths, 2020).

Given the phenotypic similarities between Sac1^ts and vap mutants (Mao, 2019), it was surprising that osbp did not affect Mys distribution or cause severe Rst accumulation. However, this is reminiscent of previous results from Drosophila neurons, where loss of Vap but not OSBP caused protein accumulation and ER stress (Moustaqim-Barrette, 2014). It is possible that, as in yeast where the presence of one out of seven OSBP homologues is sufficient for viability, OSBP functions redundantly with one or more ORPs in regulating the endosomal pathway. Indeed, CG1513, which is synthetically lethal in combination with osbp (Moustaqim-Barrette, 2014), encodes an orthologue of mammalian ORP9, which functions similarly to OSBP in sterol-PI4P exchange at ER-Golgi MCS. Alternatively, CG3860 encodes an orthologue of mammalian ORP2, which localizes to late endosomes in HeLa cells and influences sterol levels in endosomes and the PM, although countertransport of PI4P has not been shown. Mammalian ORP2 also binds ORP1L, which acts at ER-endosome MCS and promotes endosome transport, though it is unclear whether such a role is conserved in Drosophila, which lack an ORP1L orthologue. Further characterization of the Drosophila ORPs is thus needed to clarify their respective contributions to lipid homeostasis and endosomal progression (Griffiths, 2020).

Recent years have seen a proliferation of research into Sac1's roles in lipid homeostasis and the importance of PI4P regulation, as well as the development of novel probes and methods for studying phosphoinositides in vivo. This study has provided new insights into Sac1's function in protein delivery and turnover in a developing tissue, which will serve as groundwork for further investigations into the significance of Sac1 in cell physiology, organismal development, and ultimately cellular homeostasis in human health and disease (Griffiths, 2020).

While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, this study analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster. The lipidome was manipulated with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed generation of flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, ocular proteins were identified whose abundances are differentially affected by lipid saturation and unsaturation. For instance, a group of proteins was unexpectedly identified that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, two differentially lipid-responsive proteins involved in stress responses were identified, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, it was discovered that the ocular lipid class composition is robust to dietary changes, and it is proposed that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. It is anticipated that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges (Kumar, 2023).

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wg^RNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development (Raj, 2020).

Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing. Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals, and can function to repress transcription. The genome-wide studies have revealed that  ~10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters. In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways, it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila. However, the biological relevance of transcriptional pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood (Raj, 2020).

M1BP regulates retinal determination and MF progression in developing eye This study tested fthe role of transcription pausing factor, M1BP during Drosophila eye development. Downregulation of M1BP levels in the developing eye was found to result in strong suppression of eye fate, however, gain-of-function of M1BP did not affect the eye fate suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, no domain constraint in eye suppression function was seen when M1BP levels were downregulated. In addition, when M1BP levels were downregulated (ey > M1BP^RNAi) the expression of retinal determination and differentiation genes were strongly downregulated. Interestingly, it was found that protein encoded by RD genes were downregulated in ey > M1BP^RNAi background. Therefore, M1BP may not be affecting RD gene expression directly (Raj, 2020).

During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it. The two signals dpp and hh plays an important role in initiation and progression of MF. Downregulation of M1BP affects retinal differentiation as well as progression of MF. It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. This study screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc (Raj, 2020).

The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, targets were sought of M1BP transcriptional pausing function using the candidate gene approach. It was found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated. This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression. Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change when cells are treated with M1BP^RNAi (Raj, 2020).

To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BP^RNAi eye imaginal disc, this study also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGGTCACACTR) has been reported earlier. This sequence was used for MEME analysis to screen for M1BP binding sites were found in the wg gene and regulatory region (Raj, 2020).

Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development. During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc. Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development. This study found that in ey > M1BP^RNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate. Further, downregulation of wg levels, using wg^RNAi, in ey > M1BP^RNAi background rescued the eye suppression phenotype. This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Raj, 2020).

Higher levels of Wg are known to trigger developmental cell death in the developing eye field. Interestingly, in ey > M1BP^RNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene in ey > M1BP^RNAi background showed rescue of eye suppression phenotype. However, these P35 mediated rescues of ey > M1BP^RNAi were not as significant as seen with wg^RNAi. This suggests that Wg might be regulating eye fate through hth induction and eye field size by triggering caspase mediated cell death. In order to rule out that these in ey > M1BP^RNAi phenotypes are not affected by reduced cell proliferation rates, levels were tested of pH3 in these developing eye fields. It was found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye (Raj, 2020).

These results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development. However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand whether M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, the downregulation of M1BP function was studied in bi-Gal4 domains of wing imaginal disc. Whether this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc was tested. A bi-GAL4 driver which drives the expression of a transgene in wing imaginal disc was used. Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BP^RNAi) exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc. Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling. In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila (Raj, 2020).

A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5^W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis. Interestingly, the mutant allele of Spt5 (foggy^m806) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation. Therefore, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development (Raj, 2020).

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), this study revealed an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, it was shown that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations (Pasnuri, 2023).

Developmental pathways have evolved mechanisms to monitor positional information in order to generate reproducible organismal patterns. These pathways are robust and insensitive to small changes in individual processes involved. Spatial differentiation, where a population of cells undergo deterministic molecular differentiation, brings about spatial patterns. Redundancy of mechanism and negative feedback are two ways in which reliability in pattern formation is brought about. Lateral inhibition by diffusible molecules is another mechanism that can be used to generate patterns. For systems which do not depend on developmental history, environmental makeup determines their molecular differentiation contributing towards generating a pattern (Pasnuri, 2023).

This paper proposes a mechanism where relative expression levels of principal EGFR ligand, Spitz and negative feedback regulator Argos determines the extent of EGFR activation which is crucial for the periodic ommatidial pattern. The data suggests that it is not the absolute gene expression but the balance between gene networks on the whole which may contribute towards pattern formation. GMR-Gal4 is expressed in all cells posterior to the morphogenetic furrow and any expression cassette under the UAS is expressed strongly. Whereas, Elav-Gal4 is expressed only in neuronal cells and the expression is strongest towards the posterior end of the eye disc. While Elav-Gal4 expression occurs in differentiated neurons starting with R8 in the eye disc (for which EGFR signaling is not needed), but beyond R8 specification, Spitz and Argos levels are important for subsequent PR cell differentiation and also in the pupal stages. Although an equally drastic reduction was observed in argos expression when UAS-spitz dsRNA driven by GMR-Gal4 and Elav-Gal4, the eye discs show reduced dpERK staining in both cases. The reduction was lower when Elav-Gal4 driver was used, corresponding to the absence of phenotype in the adult eye. The eye discs expressing EGFRCA construct under GMR-Gal4 Gal80ts with spitz-to-argos ratio near 1, showed a discontinuous S-phase band after the morphogenetic furrow indicating a lower population of cells entering the second mitotic wave. Fewer cells entering the second mitotic wave leaves the tissue field with fewer uncommitted cells to make cell fate decisions. This can affect pattern formation to a great extent. This could also explain fewer number of bristle cells in the rough adult eyes as bristles cell fate is assigned from cells arising from the second mitotic wave. It should also be noted that the rough eye phenotype for EGFRDN is rather different from EGFRCA and shows a profusion of bristles. This mechanism of relative expression determining phenotype supports older work on the importance of spitz-to-argos dose as a critical determinant of eye patterning. Indeed mRNA levels may not be always predictive of protein levels, and both translational regulation and post-translational modifications may well affect biological function. However, under conditions of stress or where specific transcriptional programmes bring about developmental outcomes, transcript levels may be thought to be well-correlated to protein levels. In addition, experiments using smFISH allows clear identification of the cells that are expressing specific genes, where the diffusible protein end-products may not provide as conclusive answers. We did attempt performing antibody staining for Spitz and Argos but such relative measures cannot be used to comment on expression stoichiometry. This study shows a clear differential expression of spitz and argos mRNA in the early eye field contributing to photoreceptor fate determination and also addresses the sensitivity of the system to the heterogeneity in the expression levels of gene networks and makes developmental programs robust. It has to be noted of course that signaling via EGFR is not the only pathway determining the ommatidial pattern in the eye. For example, Notch is known to play an important role in the initiation of neural development and also in ommatidial rotation. Buffered regulation of genes in different developmental pathways that crosstalk can decrease sensitivity to variations in a gene network and can help explain other reproducible and stereotypical patterns generated throughout the development (Pasnuri, 2023).

Herzog (Hzg, CG5830) shares similarity to members of the haloacid dehalogenase subfamily of small CTD phosphatases. In Drosophila it is a maternal gene essential for establishment of embryonic segment polarity, and oligomerization is required for activation of phosphatase activity. While Hzg is expressed in the brain, its role has not been investigated. To that end, this study further characterized Hzg expression in the brain, and it was found to be highly expressed in neurons of the mushroom body where it localises to axons and is also expressed in cortical glia. This study investigated its role in mushroom body development as well as courtship learning and memory, but found that knockdown of Hzg had no impact on these processes. In contrast, knockdown in post-mitotic neurons in the eye resulted in disruption to ommatidial patterning and pigmentation, indicating it plays an important role in eye development (Palmer, 2023).

Lattice cells (LCs) in the developing Drosophila retina constantly move and change shape before attaining final forms. Previous studies showed that repeated contraction and expansion of apical cell contacts affect these dynamics. This study describe sa second contributing factor, the assembly of a medioapical actomyosin ring composed of nodes linked by filaments that attract each other, fuse, and contract the LCs' apical area. This medioapical actomyosin network is dependent on Rho1 and its known effectors. Apical cell area contraction alternates with relaxation, generating pulsatile changes in apical cell area. Strikingly, cycles of contraction and relaxation of cell area are reciprocally synchronized between adjacent LCs. Further, in a genetic screen, RhoGEF2 was identified as an activator of these Rho1 functions and RhoGAP71E/C-GAP as an inhibitor. Thus, Rho1 signaling regulates pulsatile medioapical actomyosin contraction exerting force on neighboring cells, coordinating cell behavior across the epithelium. This ultimately serves to control cell shape and maintain tissue integrity during epithelial morphogenesis of the retina (Rosa, 2023).

The Drosophila melanogaster eye has been instrumental for determining both how cells communicate with one another to determine cell fate, as well as cell morphogenesis and patterning. This study describes the effects of the small GTPase Rap1 on the development of multiple cell types in the D. melanogaster eye. Although Rap1 has previously been linked to RTK-Ras-MAPK signaling in eye development, this study demonstrate that manipulation of Rap1 activity is modified by increase or decrease of Delta/Notch signaling during several events of cell fate specification in eye development. In addition, it was demonstrated that manipulating Rap1 function either in primary pigment cells or in interommatidial cells affects cone cell contact switching, primary pigment cell enwrapment of the ommatidial cluster, and sorting of secondary and tertiary pigment cells. These data suggest that Rap1 has roles in both ommatidial cell recruitment/survival and in ommatidial morphogenesis in the pupal stage. They lay groundwork for future experiments on the role of Rap1 in these events (Yost, 2023). <

The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. This study shows that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. Artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens (Ghosh, 2024).

Forming the precisely curved architecture of the Drosophila corneal lens from apical ECM (aECM) requires the ZP-domain protein Dyl. Dyl acts transiently at a critical point in development to maintain the apical expansion of corneal lens-secreting cells and to assemble a scaffold containing ZP-domain proteins that acts as a convex outer boundary within which chitin and other corneal lens components are retained. These results add to a growing body of evidence that the shapes of rigid structures composed of aECM rely on specific sites of attachment to the underlying cells that are mediated by ZP-domain proteins (Ghosh, 2024).

The data show that the marked apical expansion of primary pigment cells in the pupal retina is essential to build a foundation for corneal lens assembly. Reversing this expansion either by removing Dyl or by activating myosin contraction results in severe corneal lens defects. The apicobasal contraction that occurs in dyl mutant or Mlck^CT-expressing ommatidia places the apical surfaces of cone and primary pigment cells in a more basal position than their wild-type neighbors. If secondary and tertiary pigment cells retain their attachments to the aECM, the ommatidial surface could take on a concave shape, potentially explaining the deeper inner curvature of the overlying corneal lenses. The smaller surface area of the central cells could also directly alter the shape of the corneal lens if this structure is assembled one layer at a time, like the tectorial membrane. Such a pattern of assembly would be consistent with labeling of the outer surface of the early corneal lens and the inner surface of the adult corneal lens by the chitin-binding probe, which may preferentially bind to newly deposited chitin. Constriction of the apical cell surface has been shown to produce ridges of aECM in the C. elegans cuticle and the Drosophila trachea; the results suggest that apical expansion can also drive the morphogenesis of specific aECM structures. However, not all aECM remodeling requires cell shape changes; for example, formation of sex-specific pores that accommodate chemosensory cilia in the C. elegans cuticle appears to depend on gene expression in the underlying glia rather than on the shape of these cells (Ghosh, 2024).

The notable apical constriction of cone and primary pigment cells that was observed in dyl mutants implies that Dyl is required to maintain their expanded shape. Dyl is located on the apical surfaces of these cells, and it and other ZP-domain proteins have been shown to attach the plasma membrane to the aECM. These attachments can alter cell shape; for instance, apical expansion of cells in the pupal wing is dependent on the ZP-domain proteins Miniature and Dusky; NOAH-1 and NOAH-2 are required for the concerted cell shape changes that drive elongation of the C. elegans embryo , and Hensin is needed to convert flat β- to columnar α-intercalated cells in the kidney. The NOAH-1, NOAH-2, and FBN-1 ZP-domain proteins in the embryonic sheath of C. elegans resist deformation of the epidermis by mechanical forces. It is suggested that attachment to the aECM by Dyl enables cone and primary pigment cells to resist mechanical tension exerted by the actomyosin cytoskeleton. Active myosin and βH-spectrin accumulate in dyl mutant cells, and reducing the level of these components partially rescues apical constriction. However, these proteins appear disorganized and do not form foci like those seen in the wild-type cells. Similar foci are associated with changes in apical cell area driven by pulsatile constriction and ratcheting, but continuous constriction can occur with a diffuse apical actomyosin distribution, for instance, in boundary larval epithelial cells. The loss of apical anchorage may cause dyl mutant cells to constrict in a continuous, passive manner that does not involve controlled ratcheting (Ghosh, 2024).

Although electron micrographs suggest that Dyl establishes a physical link between the apical plasma membrane and the aECM, this role at contact sites is surprising because it is likely that the extracellular domain of Dyl is proteolytically cleaved from its transmembrane domain. Overexpressed Dyl appears to diffuse away from the cells that express it (Adler, 2013; Nagaraj, 2012), and Dyl contains a putative furin cleavage site that has been used to enable secretion of a reporter protein. Nevertheless, loss of dyl has cell-autonomous effects on apical constriction and chitin accumulation in the retina, indicating that the protein does not freely diffuse in this tissue and may associate with other transmembrane proteins and/or with immediately overlying aECM. Alternative explanations for Dyl function are also possible. Rather than directly attaching the membrane to the matrix, Dyl might affect the properties of the developing aECM, for instance, by endowing it with sufficient tensile strength to counteract cytoskeletal forces that would otherwise drive apical constriction of the cone and pigment cells (Ghosh, 2024).

The data also reveal a role for other ZP-domain proteins, including Dpy and Pio, in establishing an external scaffold that helps to shape the corneal lens. Although dpy mutant ommatidia show little constriction, indicating that dpy acts downstream of or in parallel to the cell shape changes, they fail to accumulate chitin at the normal time and produce deformed corneal lenses. As chitin is not found trapped inside dyl or dpy mutant cells, it is likely that it diffuses away in the absence of the scaffold. Pio is not necessary to retain chitin at this stage, but it appears to have an analogous role later in development, when it is present in the pseudocone, to maintain the normal curved boundary of the inner surface of the corneal lens. Dpy and Pio have a similar function in the trachea, where they provide an elastic structural element of the lumen that controls its shape and size and maintains the chitin matrix. Removal of this structure by proteolysis is necessary for subsequent gas filling of the airway, and it is possible that the external corneal lens scaffold is likewise proteolytically degraded in late pupal stages. Transient aECM structures containing ZP-domain proteins also shape many cuticular and tubular structures in C. elegans, and α-tectorin on the surface of supporting cells templates the assembly of collagen fibrils in each layer of the tectorial membrane (Ghosh, 2024).

The arrangement of Dpy and Pio is dependent on Dyl; in wild-type ommatidia, they form linear structures, but this organization is not apparent in dyl mutants.The precise nature of these structures is not known, nor what additional components they may contain. ZP-domain proteins are capable of forming heteropolymeric filaments, and another ZP protein, Qsm, is known to promote the secretion and remodeling of Dpy filaments that link tendon cells to the pupal cuticle, increasing their tensile strength. Because Dyl is only present in the retina for a short time, it may be required to initiate the assembly of a scaffold structure that then becomes self-sustaining. Alternatively, because apical constriction is sufficient to alter Dpy organization and delay chitin accumulation, the role of Dyl may be limited to maintaining apical expansion. Nevertheless, the requirement for dyl for normal chitin deposition in bristles and wing hairs suggests that promoting chitin assembly, directly or indirectly, is one of its primary functions (Ghosh, 2024).

Other components of the corneal lens may also be affected by loss of dyl. Although chitin levels appear normal in sections of adult retinas, scanning electron micrographs show that the external surface of the corneal lens is incomplete. Induced apical constriction does not fully reproduce this loss of surface structure, suggesting that it reflects an independent function of Dyl. In the pupal wing, dyl belongs to a cluster of genes with peak expression at 42 hours APF, when the outer envelope layer of the cuticle is being deposited, but it influences the structure of inner layers as well. dyl expression in the embryo is also limited to a short time window in which apical cell remodeling and initial cuticle deposition occur (Fernandes, 2010). Another gene expressed at this time point, tyn, is required for the barrier function of the embryonic envelope layer. It is not clear whether the corneal lens has a typical cuticular envelope, but its outermost layer forms nanostructures composed of the Retinin protein and waxes. Although retinin mRNA is not strongly expressed until very late in pupal development, its expression is initiated at mid-pupal stages when Dyl is present. It is possible that Dyl and the Dpy-Pio scaffold also control the retention of Retinin and other components of the outer layer (Ghosh, 2024).

In summary, this study shows that the development of a biconvex corneal lens depends on both its upper and lower boundaries. The upper boundary, a convex shell of aECM that contains the ZP-domain proteins Dpy and Pio, encloses chitin and its associated proteins as they are secreted by cone and pigment cells. The shape of this shell may be defined by its attachment to the peripheral lattice cells and the pressure exerted on its center by secreted corneal lens components. The lower boundary is the apical plasma membrane of the cone and primary pigment cells. This membrane is flat until pseudocone secretion initiates late in pupal development, and the phenotype of dyl mutants suggests that it is maintained taut and expanded by attachment to the aECM. Loss of this expansion or of the pseudocone components Dpy or Pio gives the inner surface of the corneal lens a deeper curvature. It would be interesting to investigate whether mechanical forces exerted on the stromal ECM of the human cornea, perhaps through the ZP-domain proteins ZP4 and endoglin present in the underlying corneal endothelium, affect its shape and refractive power (Ghosh, 2024).

Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. This study reports that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, these data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context (Zhang, 2023).

The highly conserved Target of Rapamycin Complex 1 (TORC1) is a central regulator of growth and metabolism in eukaryotes. In the presence of positive upstream inputs, TORC1 promotes anabolic metabolism and growth by phosphorylating numerous downstream targets including S6K and 4EBP. Conversely, when exposed to negative cues, such as limited nutrients, or the lack of growth factors, cells downregulate TORC1 to activate catabolic metabolism and inhibit growth. The deregulation of TORC1 is implicated in a wide array of diseases including cancer, epilepsy, and aging. Thus, there is intense interest in obtaining a mechanistic understanding of how upstream signaling pathways regulate TORC1 activity (Zhang, 2023).

The Rag GTPase is a heterodimer comprised of a Rag A/B subunit and a RagC/D subunit, that recruits TORC1 to lysosomes for activation by the small GTPase Rheb, when the RagA/B component of the GTPase is in the GTP-bound state. The GTPase-activating protein toward Rags (GATOR) complex is an important upstream regulator of TORC1 that responds to the presence of nutrients. The GATOR complex, originally identified in yeast and named the Seh1 Associated (SEA) complex, is comprised of two subcomplexes, GATOR1 and GATOR2. The GATOR1 complex, which contains the proteins Nprl2, Nprl3, and DEPDC5/Iml1, inhibits TORC1 activity by serving as a GAP (GTPase-activating protein) for the lysosomally located Rag GTPase (Zhang, 2023).

The GATOR2 complex inhibits GATOR1 and thus serves to activate TORC1. However, the mechanism by which GATOR2 inhibits GATOR1 remains unknown. In its initial functional characterization in mammalian and Drosophila cultured cells, the GATOR2 complex was reported to contain five protein Mios/Mio, Seh1, Sec13, Wdr24, and Wdr59. In these studies, knockdowns of GATOR2 components resulted in the constitutive activation of GATOR1 and decreased TORC1 activity. Similarly, Drosophila mutants of the GATOR2 components Mio, Seh1, and Wdr24 exhibit decreased TORC1 activity and growth in the female germline. However, a recent study from Schizosaccharomyces pombe reported that SEA3/WDR59 inhibits TORC1 activity as a component of the GATOR1 complex. Notably, this is oppositive to the role assigned to Wdr59 based on studies in both human and Drosophila cultured cells. Additionally, deletions of Wdr59 in HEK293 cells and mouse embryonic fibroblasts result in a partial resistance to nutrient deprivation. Thus, the exact function of Wdr59 within the GATOR-TORC1 signaling pathway remains unclear (Zhang, 2023).

This study defines the in vivo requirement for the GATOR component Wdr59 in Drosophila. Wdr59 displays two distinct functions depending on cell type. First, in the ovary and the eye imaginal disc brain complex, Wdr59 acts upstream of the GATOR2 complex to inhibit TORC1 activity. Second, in the adult fat body and mammalian HeLa cells, Wdr59 protects the GATOR2 complex from proteolysis and promotes TORC1 activity. These data provide mechanistic insight into the complex role of the GATOR component Wdr59 in the tissue-specific regulation of TORC1 activity (Zhang, 2023).

The GATOR complex is an essential upstream regulator of TORC1 that is frequently mutated in human disease. This study describes a dual function for the GATOR component Wdr59 in TORC1 regulation in Drosophila. Surprisingly, Wdr59 functions were found to inhibit or promote TORC1 activity depending on cellular context. These studies broaden understanding of the GATOR-TORC1 signaling axis in metazoans and demonstrate the importance of examining metabolic regulation in vivo (Zhang, 2023).

In vivo studies often reveal tissue-specific and/or metabolic requirements for genes that are not observed in cell culture. In cultured cells from both mammals and Drosophila, RNAi depletions of wdr59 result in decreased TORC1 activity and slow growth, thus phenocopying depletions/knockouts of the GATOR2 components wdr24, seh1, and mio. Based on these initial studies, Wdr59 was assigned to the GATOR2 complex, which promotes TORC1 activity by downregulating the TORC1 inhibitor GATOR1. However, in vivo studies indicate wdr59 is not obligate member of the GATOR2 complex. Using Drosophila as a model, this study found that wdr59 mutant ovaries have increased TORC1 activity and growth rates and an attenuated response to nutrient stress. Notably, these phenotypes are the opposite of those reported for mutants of the GATOR2 components mio, seh1, and wdr24, but phenocopy those observed in mutants of the GATOR1 components nprl2, nprl3, and iml1. Thus, in the Drosophila ovary, Wdr59 functions to restrict TORC1 activity. In line with these findings, the binding of RagAGTP to the Nprl2 component of the GATOR1 complex was demonstrated to be decreased in wdr59 mutants, again the opposite of what was observed in mutants of the GATOR2 component wdr24. Taken together, these data suggest that wdr59 inhibits TORC1 activity by increasing the activity of the TORC1 inhibitor GATOR1. Notably, recent evidence from s. pombe proposes that Wdr59, known as SEA3 in yeast, acts to inhibit TORC1 as a component of the GATOR1 complex. However, epistasis analysis indicated that in Drosophila, Wdr59 is not a component of the GATOR1 complex but instead functions as an inhibitor of GATOR2 (Zhang, 2023).

How might wdr59 impact TORC1 activity? Biochemical, structural, and computational analysis from yeast indicate that SEA3/wdr59 is well positioned to function as an interacting hub connecting GATOR2 with GATOR1. Consistent with this observation, this study found that, as is observed in fission yeast, wdr59 regulates the interaction between components of the GATOR1 and GATOR2 complexes, with an increased association of GATOR2 with GATOR1 observed in the wdr59 mutant background. Thus, one possible model to explain these data is that wdr59 inhibits TORC1 activity by restricting the interaction of the GATOR2 complex with GATOR1, thus unleashing its GAP activity and TORC1 inhibitory potential. A prediction from this model is that components of the GATOR2 complex will be epistatic to Wdr59. In other words, Wdr59 requires the presence of an active GATOR2 complex to regulate TORC1 activity. Consistent with this prediction, it was fond that in the Drosophila ovary, wdr59, GATOR2 double mutants have a GATOR2-like phenotypes with reduced growth and TORC1 activity, strongly suggesting that GATOR2 is downstream of Wdr59. In contrast, in mutants or depletions of the GATOR1 components nprl2, nprl3, and Iml1, RagA remains in its TORC1-activating GTP-bound state, promoting the constitutive recruitment and activation of TORC1 on lysosomes independent of the status of GATOR2. Thus, GATOR1 components, unlike Wdr59, are epistatic to upstream members of the pathway including components of the GATOR2 complex. Taken together these data support the model that in Drosophila, Wdr59 is not required for GATOR1 function but instead acts upstream of the complex to regulate the activity of the GATOR1 inhibitor GATOR2. Importantly, the data demonstrate that the GATOR2 complex can inhibit GATOR1 independent of the Wdr59 subunit (Zhang, 2023).

Currently, there are conflicting reports on the role of Wdr59/Sea3 in the regulation of TORC1 activity. In Drosophila and mammalian cultured cells, as well as in mouse breast cancer tumors, Wdr59 promotes TORC1 activity while in fission yeast Wdr59 inhibits TORC1 activity. The current results provide a potential explanation for this contradiction. In Drosophila, Wdr59 either promotes or inhibits TORC1 activity depending on cell type. In the female germline and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity. However, in the adult fat body, Wdr59 promotes TORC1 activity. Importantly, in the adult fat body, but not the ovary, Wdr59 is required for the accumulation of the Mio protein. Thus, a possible reason that wdr59 mutant fat bodies have reduced TORC1 activity is that they do not have a functional GATOR2 complex. This model is consistent with the finding that the GATOR2 complex is downstream of Wdr59 (Zhang, 2023).

To further explore why Wdr59 promotes TORC1 activation in some cellular contexts, the levels of GATOR2 proteins were examined in Wdr59KO HeLa cells. It was wondered if the TORC1 promoting function for Wdr59 observed in cultured cells might reflect the requirement for Wdr59 protein to accumulate components of the GATOR2 complex. Indeed, dramatically lower levels of the GATOR2 components Mios and Wdr24 were found in Wdr59KO HeLa cells due to proteolytic destruction by the proteosome. Strikingly, inhibiting the proteasome pharmacologically resulted in increased levels of GATOR2 proteins which was accompanied by a partial rescue of TORC1 activity. These data strongly suggest that the low TORC1 activity observed in Wdr59KD or Wdr59KO HeLa cells is due at least in part to the concomitant decrease in GATOR2 protein levels (Zhang, 2023).

Taken together, these data support the model that Wdr59 either promotes or inhibits TORC1 activity depending on cellular context. The GATOR-TORC1 signaling pathway is frequently cited as a potential target of pharmaceutical intervention because of its role in cancer and epilepsy. Thus, it is essential to have a full mechanistic understanding of the in vivo function of the GATOR complex in the regulation of TORC1 signaling and growth in metazoans (Zhang, 2023).

Prior to submission of this manuscript, a detailed cryo-electron microscopy structure of the human GATOR2 complex was published. It was reported that GATOR2 is a large 1.1 Mda complex that forms a cage-like structure, built on a continuous scaffold. As previously described, GATOR2 complex components contain numerous features common to membrane coating complexes which can form scaffolds that alter the curvature of membranes. Consistent with these studies, the authors report that the two copies of the Wdr59 subunit mediate the association of GATOR2 with GATOR1. However, as discussed above, it was found that Wdr59 opposes the association of GATOR2 with GATOR1 in several Drosophila tissues, the opposite of what is reported in this study in HEK293T cells. Whole animal studies in Drosophila have determined that there are unique tissue-specific requirements for multiple individual GATOR2 subunits, including Mio, Seh1, Wdr24, and now Wdr59. Going forward, it will be fascinating to determine how GATOR2 structure mediates tissue-specific GATOR2 functions in vivo (Zhang, 2023).

Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. This study identified the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). These findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, this study identified the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands understanding of NF-YC's role from cell cycle control to encompass developmental processes (Avellino, 2023).

The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the Endoplasmic Reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, a dominant modifier screen was performed of the 3rd chromosome for enhancers and suppressors of this Jagn RNAi-induced rough eye phenotype. this study screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn RNAi phenotype. Based on the functions of the genes covered by the deficiencies, genes were identified that displayed a suppression or enhancement of the Jagn RNAi phenotype. These include Division Abnormally Delayed (Dally), an heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on current understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis (Ascencio, 2023).

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina (Muliyil, 2020).

This paper reports a previously unrecognised role of ADAM17 and TNF in protecting Drosophila retinal cells from age- and activity-related degeneration. Loss of ADAM17 and TNF signalling in retinal glial cells causes an abnormal accumulation of LDs in young glial cells. These LDs disperse by about 2 weeks after eclosion (middle age for flies), and their loss coincides with the onset of severe glial and neuronal cell death. By 4 weeks of age, no intact glia or neurons remain. Cell death depends on neuronal activity: retinal degeneration and, to a lesser extent, LD accumulation are rescued in flies reared fully in the dark. LD accumulation does not merely precede, but is actually responsible for subsequent degeneration, because preventing the accumulation of LDs fully rescues cell death. The data indicate that Eiger/TNF released by ADAM17 acts specifically through the Grindelwald TNF receptor. Loss of ADAM17-mediated TNF signalling also leads to elevated production of mitochondrial ROS in glial cells, causing activation of the JNK pathway and elevated lipogenic gene expression. Together, these changes trigger cell death through the production of toxic peroxidated lipids. Importantly, toxicity is also contributed to by ROS generated by normal activity of neighbouring neurons. Finally, this study shows that a similar signalling module is conserved in mammalian cells: when ADAM17 is inhibited in human iPSC-derived microglial-like cells, the same series of events is seen: LD accumulation, elevated mitochondrial ROS and high levels of toxic peroxidated lipids (Muliyil, 2020).

It is proposed that TNF is an autocrine trophic factor that protects retinal pigmented glial cells from age-related cumulative damage caused by the ROS that are normal by-products of neuronal activity. This ADAM17/TNF protection system is located specifically in retinal glial cells, but its role is to protect both glia and neighbouring neurons. In the absence of this TNF cytoprotective pathway, severe early-onset retinal neurodegeneration is seen. The data imply that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs disperse. This occurs in Drosophila middle age, when LDs stop accumulating and begin to disperse, triggering the cytotoxic phase of the ADAM17-/- phenotype. It is important to emphasise that despite the ADAM17/TNF protection system being located specifically in retinal glial cells, there is neuronal involvement. Not only does TNF indirectly protect against neurodegeneration, but photoreceptor neurons are also significant sources of the ROS that generate the toxic peroxidated lipids in glia. More generally, this work provides a model for investigating more widely the functional links between ageing, cellular stress, lipid droplet accumulation and neurodegeneration. Indeed, in the light of the discovery that the pathway discovered in Drosophila is conserved in human microglia-like cells, it is significant that lipid droplets have been reported to accumulate in human microglia, cells that are increasingly prominent in the pathology of Alzheimer's disease and other neurodegenerative conditions (Muliyil, 2020).

In a Drosophila model of neuronal mitochondrionopathies, abnormal neuronal ROS production led to elevated neuronal lipid production, followed by transfer of the lipids to the PGCs, where LDs accumulated. In that work, lipase expression in neurons suppressed LD accumulation; in contrast, suppression was only observed when lipase was expressed in PGCs, not neurons, suggesting that in the case of ADAM17 mutants, the primary source of accumulating lipids is the glial cells. Despite not being able to detect a role for neuronal lipid production in ADAM17-/- mutants, it was found that photoreceptor neurons are significant sources of the ROS that generate the toxic peroxidated lipids in glia. Despite these differences between this work and what has been previously reported, a growing body of work points to a close coupling between ROS, the JNK pathway, lipid droplets and cellular degeneration, a relationship conserved in mammals. This study did not investigate the involvement of SREBP in mediating LD accumulation caused by ADAM17 loss, but its well-established connection with stress-induced and JNK-mediated lipid synthesis suggests that it is a likely additional shared component of this conserved regulatory axis (Muliyil, 2020).

It has become clear that LDs are much more than simply passive storage vessels for cellular lipids; they have multiple regulatory functions. Indeed, although this study highlighted a developing picture of an LD/ROS-dependent trigger of cell death, in other contexts LDs have protective functions against oxidative damage, both in flies and mammals. This may occur by providing an environment that shields fatty acids from peroxidation by ROS and/or by sequestering toxic peroxidated lipids. Although this superficially appears to contradict the theme of LD/ROS toxicity, it is important to recall that in LD-related cell death is not simultaneous with LD accumulation. In fact, degeneration temporally correlates with the dispersal of LDs in middle age, rather than their earlier accumulation. Together, the strands of evidence from several studies suggest that it is the combination of elevated ROS and the dispersal of abnormally high quantities of lipids from previously accumulated LDs that trigger death. This suggests that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs break down in the presence of high levels of ROS. Experiments with Brummer lipase are consistent with this idea: the Brummer lipase was expressed from early in development, thereby preventing abnormal LD accumulation, and this protected against cell death. This sequence of events implies the existence of a metabolic switch, when LDs stop accumulating and begin to disperse, triggering the toxic phase of ADAM17 loss. It will be interesting in the future and may provide insights into the normal ageing process, to understand the molecular mechanism of this age- and/or activity-dependent change (Muliyil, 2020).

ADAM17 is one of the most important shedding enzymes in humans, responsible for the proteolytic release of a vast array of cell surface signals, receptors and other proteins. Because of its role in signalling by both TNF and ligands of the EGF receptor, it has been the focus of major pharmaceutical efforts, with a view to treating inflammatory diseases and cancer. It is therefore surprising that it has been very little studied in Drosophila. This is the first report of Drosophila ADAM17 mutants. This study also confirmed for the first time that Drosophila ADAM17 is indeed an active metalloprotease, able to shed cell surface proteins including the Drosophila TNF homologue Eiger. The only other description of Drosophila ADAM17 function is mechanistically consistent with the data, despite relating to a different physiological context. In that case, ADAM17 was shown to cause the release of soluble TNF from the fat body so that it can act as a long range adipokine (Agrawal, 2016). Other ADAM17 substrates in different developmental or physiological contexts cannot be ruled out, although the relatively subtle phenotype of null mutant flies implies that ADAM17 does not have essential functions that lead to obvious defects when mutated. Moreover, wdevelopmental defects or LD accumulation were not observed in any neuronal or non-neuronal ADAM17-/- larval tissues, suggesting that the mechanism reported in this study is both age and tissue specific (Muliyil, 2020).

Although TNF is sometimes viewed as a specific cell death-promoting signal, and the pathways by which it activates caspase-induced apoptosis have been studied extensively in flies and mammals, the response to TNF is in fact very diverse, depending on the biological context. Indeed, its most well-studied role in mammals is as the primary inflammatory cytokine, released by macrophages and other immune cells, and triggering the release of other cytokines, acting as a chemoattractant, stimulating phagocytosis, and promoting other inflammatory responses. However, TNF has not previously been shown to have trophic activity, protecting cells in the nervous system from stress-induced damage, although a link with the Nrf2/Keap1 redox pathway in cardiomyocytes provides an interesting parallel (Muliyil, 2020).

In conclusion, this work highlights three important biological concepts. The first is to identify a new function for the ADAM17/TNF pathway in a cytoprotective role that protects Drosophila retinal cells against age- and activity-dependent degeneration. This contrasts with its well-established roles in inflammation and cell death. Secondly, the existence is highlighted of a glia-centric cellular pathway by which the breakdown of accumulated LDs and ROS together participate in promoting stress-induced and age-related cell death. Finally, this study has shown that the core phenomenon of the ADAM17 protease, acting to regulate the homeostatic relationship between ROS and LD biosynthesis, is conserved in human microglial cells, which themselves are intimately involved in neuroprotection (Muliyil, 2020).

The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. This study used Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit (see Nervana 1 and Nervana 2), partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration (Luan, 2014).

The Na+/K+-ATPase transports Na+ and K+ against their concentration gradients across the cell membrane to maintain a low Na+ and high K+ concentration within the cells. These ion gradients determine the resting membrane potential and form the basis of the excitability of neurons. The Na+ gradient also provides the driving force for various secondary active transporters that import glucose, amino acids, and other nutrients into the cell. Additionally, the ion concentrations maintained by Na+/K+-ATPase are important for regulating cellular volume and preventing cells such as neurons from swelling and lysing (Luan, 2014).

Considering the importance of Na+/K+-ATPase in basic cellular functions, it is not surprising that either dysregulation or deficiency of neuronal Na+/K+-ATPase was observed in many neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and rapid-onset dystonia Parkinsonism (RDP). Thus, disrupting normal Na+/K+-ATPase activity in neurons has been proposed to contribute to the pathogenesis of neurodegeneration. Nevertheless, the link between the disrupting neuronal Na+/K+-ATPase activity and neuronal dysfunction/degeneration has yet to be clarified (Luan, 2014).

Na+/K+-ATPase is composed of at least two subunits: a large catalytic α subunit and a regulatory, single-transmembrane-domain β subunit. Mammals have three α-subunit and two β-subunit genes and may express six structurally distinct Na+/K+-ATPase isoforms. In the brain, although the α3 and β2 subunits are expressed predominantly in neurons, the α2 and β1 subunits are found primarily in glia, and the α1 subunit is ubiquitously expressed. The Na+/K+-ATPase in glia is required to maintain a low K+ level in the neuronal environment and thus has a large impact on neuronal function and survival. Na+/K+-ATPase inhibitors like ouabain act on all Na+/K+-ATPase isoforms and cannot differentiate between the cell-autonomous effects of the Na+/K+-ATPase in neurons from those derived from the neighboring glia. Thus, genetic approaches are needed to modulate the Na+/K+-ATPase level in neurons to investigate the function of neuronal Na+/K+-ATPase. Genetic studies on the impact of neuronal Na+/K+-ATPase deficiency in the past decade, which were mostly based on characterization of heterozygous mutant mice of the α3 subunit, have identified defects in the function of central brain neurons but have not provided direct evidence of neurodegeneration (Luan, 2014).

The Drosophila visual system expresses only one type of α subunit, ATPα, and three β subunits, Nrv1–3. This study used Drosophila photoreceptors as a genetic model to study the cell-autonomous functions of neuronal Na+/K+-ATPase. Although ATPα mutants in Drosophila exhibit extensive neurodegeneration, these mutants were not used because the degeneration is due to the loss of Na+/K+-ATPase not only in neurons but also in neighboring non-neuronal cells. Instead, using a UAS/Gal4-mediated RNAi approach, knocked down ATPα and Nrv1-3 were generated specifically in photoreceptors, and the impact of this knockdown was assessed on visual signaling and photoreceptor integrity in the fly (Luan, 2014).

Na+/K+-ATPase activity establishes and maintains the characteristic transmembrane gradients of Na+ and K+, which underlie essentially all vertebrate and invertebrate cellular physiology. Although the importance of Na+/K+-ATPase to the function and survival of both neurons and non-excitable cells has been demonstrated by decades of pharmacological studies, the involvement of neuronal Na+/K+ ATPase defects in neurodegeneration has yet to be demonstrated in vivo. This cell-specific RNAi study reveals for the first time that Na+/K+-ATPase is essential for normal neuronal function of Drosophila photoreceptors. More importantly, this work provides in vivo evidence that links neuronal Na+/K+-ATPase deficiency to age-dependent neurodegeneration (Luan, 2014).

Genetic studies on neuronal Na+/K+-ATPase function in the past decade, which were primarily based on the characterization of mice heterozygous for a mutation of the α3 subunit, have focused on central brain neurons; however, the importance of functional Na+/K+-ATPase in sensory neurons remains largely unknown. This study shows that knockdown of ATPα in Drosophila photoreceptor neurons abolishes their response to light, resulting in complete blindness in the fly. These results confirm the importance of Na+/K+-ATPase in animal sensory functions. The light response of Drosophila photoreceptors is mediated by cation influx (mostly Na+) through light-stimulated TRP channels. Photoreceptors in ATPα knockdown flies were unresponsive to light for two reasons. First, without a sufficient K+ gradient across the cell membrane, the cell has no negative resting membrane potential and, thus, no electrical driving force for cation influx through TRP channels. Second, in the absence of Na+/K+-ATPase, photoreceptors may have already accumulated a high intracellular level of Na+ in the dark, which prevents extracellular Na+ flow into the cell through TRP channels during light stimulation. However, loss of the light response may not be attributed to morphological defects in the photoreceptor. First, temporally controlled knockdown of ATPα in the adult stage excludes the involvement of obvious developmental problems. Second, the light response of photoreceptors was abolished in 1-day-old ATPα knockdown flies despite the overall normal shape of the rhabdomeres. Based on these findings, it is concluded that loss of the light response is independent of degeneration in ATPα knockdown photoreceptors (Luan, 2014).

Drosophila photoreceptors have been used as a genetic model for retinal degeneration studies. Photoreceptor degeneration in many mutants is caused by defects in the regulation of the visual transduction cascade and is light-dependent. In ATPα knockdown photoreceptors, however, the visual cascade may not mediate or regulate degeneration because light deprivation does not change the severity of neurodegeneration in 10-day-old flies. Instead, the progressive degeneration in ATPα knockdown photoreceptors has demonstrated characteristics of apoptotic/necrosis hybrid cell death that are reminiscent of those observed in Na+/K+-ATPase-inhibited mammalian cells. When Na+/K+-ATPase is inhibited by ouabain, mammalian cells undergo hybrid cell death, which has been attributed to a loss of intracellular K+ ions. In vitro studies suggest that the depletion of intracellular K+ may induce apoptosis or act as a necessary cofactor to promote apoptosis. It is estimated that the intracellular levels of K+ in ATPα knockdown photoreceptors could be as low as 10 mM, which is comparable to the 50%-80% decrease in K+ observed in ouabain-treated mammalian cells. Thus, the low levels of intracellular K+ could have contributed to the degeneration of ATPα knockdown photoreceptors. Additionally, Ca2 + overload may also have a role in photoreceptor degeneration. The depolarization of the membrane potential in ATPα knockdown photoreceptors may activate voltage-gated Ca2 + channels and a reversed operation of the Na+-Ca2 + exchanger. Both activities will increase the intracellular Ca2 + concentration and could promote necrotic cell death. Finally, accumulation of Na+ inside ATPα knockdown photoreceptors impairs the driving force of nutrient import through the secondary membrane transporters, which may also play a role in cell degeneration. Hybrid cell death, an intermediate form of cell death falling along an apoptosis-necrosis continuum, can also be found in the neurodegeneration caused by excitotoxicity and ischemia. Therefore, further studies on the role Na+/K+-ATPase in hybrid cell death will elucidate the mechanism of the cell death bearing both apoptotic and necrotic features in different neuropathological conditions (Luan, 2014).

Until now, most in vivo studies on Na+/K+-ATPase-related neurodegeneration have relied on either pharmacological agents (Bignami, 1966 and Lees, 1994) or Drosophila ATPα mutants. Those studies have suggested that both dysregulation and deficiency of Na+/K+-ATPase lead to extensive neurodegeneration. However, the degeneration observed in those studies could be partially derived from defects in non-neuronal tissues and cells in the brain. For example, Na+/K+-ATPase in the blood-brain-barrier participates in the maintenance of water and ion homeostasis in the central nervous system (CNS), which is critical for neuronal function and survival. In the Drosophila auditory organ (Johnston's organ), Roy found that knocking down ATPα in scolopale cells, principal support cells that enclose neuronal dendrites, results in neuronal dysfunction and complete deafness. Additionally, a defect of Na+/K+-ATPase in astrocytes could be responsible for neonatal seizures and spongiform encephalopathy. In common neurodegenerative disorders such as AD, PD and RPD, however, Na+/K+-ATPase is only reduced in specific subgroups of neurons. To better mimic the neuropathological conditions of neurodegenerative diseases to study this degeneration mechanism, it is necessary to specifically downregulate Na+/K+-ATPase in particular neurons to avoid perturbations in other cells (Luan, 2014).

Because homozygous mutations in the mouse α3 subunit of Na+/K+-ATPase cause neonatal lethality, genetic studies on neuronal isoforms of Na+/K+-ATPase have so far been primarily based on the characterization of heterozygous α3 mutants. Those mouse studies, however, have not revealed direct evidence of neurodegeneration in the brain most likely due to the relatively moderate reduction of Na+/K+-ATPase activity in the heterozygous mutants. This in vivo model using Drosophila photoreceptors, which mimics the neuropathological conditions of those neurodegenerative disorders, could be a valuable tool for further investigating the mechanism of neuronal Na+/K+-ATPase deficiency-mediated neurodegeneration. In addition to the genetic tools for gene modulation, Drosophila utilizes simple assays such as ERG, phototaxis and optomotor responses, and the optical neutralization assay to evaluate both the function and morphology of photoreceptor neurons. The loss of Na+/K+-ATPase in photoreceptors does not change the environmental K+ levels, allowing the study the cell-autonomous effects of neuronal Na+/K+-ATPase deficiency. Taking advantage of this simple and convenient neuronal model may allow identification of the key players in Na+/K+-ATPase deficiency-mediated neurodegeneration, which will thereby guide in the design of new therapeutic strategies for neurodegenerative disorders (Luan, 2014).

This study has provided evidence that either dysregulation or deficiency of neuronal Na+/K+-ATPase causes abnormal depolarization of neurons by disrupting the intracellular ion balance instead of extracellular ion homeostasis, which leads to neuronal dysfunction and behavioral abnormality. Furthermore, disrupted neuronal Na+/K+-ATPase activity triggers progressive neurodegeneration. Therefore, this study suggests that early intervention against dysregulation or deficiency of neuronal Na+/K+-ATPase may alleviate the progression of neurodegenerative disorders (Luan, 2014).

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to beta-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, CG9317, an SLC22 transporter family protein, was identified and named CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals (Xu, 2015).

Drosophila photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. This anatomical arrangement in larger flies suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test the hypothesis, responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators were measured. Data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. Decreased activity in photoreceptors was observed in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, these results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing (Weir, 2016).

Spectral information is commonly processed in the brain through generation of antagonistic responses to different wavelengths. In many species, these color opponent signals arise as early as photoreceptor terminals.This study measured the spectral tuning of photoreceptors in Drosophila. In addition to a previously described pathway comparing wavelengths at each point in space, A horizontal-cell-mediated pathway similar to that was found found in mammals. This pathway enables additional spectral comparisons through lateral inhibition, expanding the range of chromatic encoding in the fly. Together, these two pathways enable efficient decorrelation and dimensionality reduction of photoreceptor signals while retaining maximal chromatic information. A biologically constrained model accounts for the findings and predicts a spatio-chromatic receptive field for fly photoreceptor outputs, with a color opponent center and broadband surround. This dual mechanism combines motifs of both an insect-specific visual circuit and an evolutionarily convergent circuit architecture, endowing flies with the ability to extract chromatic information at distinct spatial resolutions (Heath, 2020).

More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although it is now known that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. This study reverse-engineered the neural code of Drosophila photoreceptors and shows for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. To mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli (Friederich, 2016).

Holometabolous insects, like fruit flies, grow primarily during larval development. Scarce larval feeding is common in nature and generates smaller adults. Despite the importance of vision to flies, eye size scales proportionately with body size, and smaller eyes confer poorer vision due to smaller optics. Variable larval feeding, therefore, causes within-species differences in visual processing, which have gone largely unnoticed due to ad libitum feeding in the lab that results in generally large adults. Do smaller eyes have smaller ommatidial lenses, reducing sensitivity, or broader inter-ommatidial angles, reducing acuity? And to what extent might neural processes adapt to these optical challenges with temporal and spatial summation? To understand this in the fruit fly, a distribution of body lengths (1.67-2.34mm; n=24) and eye lengths (0.33-0.44mm; n=24) was generated, resembling the distribution of wild-caught flies, by removing larvae from food during their third instar. Smaller eyes have substantially fewer and smaller ommatidia separated by slightly wider inter-ommatidial angles. This corresponds to a greater loss in contrast sensitivity than spatial acuity. Using a flight arena and psychophysics paradigm, it was found that smaller flies lose little spatial acuity, and recover contrast sensitivity by sacrificing temporal acuity at the neural level. Therefore, smaller flies sacrifice contrast sensitivity to maintain spatial acuity optically, but recover contrast sensitivity, almost completely, by sacrificing temporal acuity neurally (Currea, 2018).

The Drosophila retina contains light-sensitive photoreceptors (R cells) with distinct spectral sensitivities that allow them to distinguish light by its spectral composition. R7 and R8 photoreceptors are important for color vision, and can be further classified into pale (p) or yellow (y) subtypes depending on the rhodopsin expressed. While both R7y and R7p are sensitive to UV light, R8y and R8p detect light in the green and blue spectrum, respectively. The ability of R7 and R8 photoreceptors to distinguish different spectral sensitivities and the natural preference for Drosophila towards light sources (phototaxis), allow for the development of a phototactic T-maze assay that compares the functionality of different R7 and R8 subtypes. A "UV vs. blue" choice can compare the functionalities of R7p and R8p photoreceptors, while a "UV vs. green" choice can compare the functionalities of R7y and R8y photoreceptors. Additionally, a "blue vs. green" choice could be used to compare R8p and R8y photoreceptors, while a "dark vs. light" choice could be used to determine overall vision functionality. The phototactic T-maze assay presented in this study is a robust, straight-forward and an inexpensive method to study genetic and developmental factors that contribute to the individual functionality of R7 and R8 photoreceptors, and is especially useful when performing large-scale genetic screens (Shaw, 2020).

Color vision is an important sensory capability of humans and many animals. It relies on color opponent processing in visual circuits that gradually compare the signals of photoreceptors with different spectral sensitivities. In Drosophila, this comparison begins already in the presynaptic terminals of UV-sensitive R7 and longer wavelength-sensitive R8 inner photoreceptors that inhibit each other in the medulla. How downstream neurons process their signals is unknown. This study reports that the second order medulla interneuron Dm8 is inhibited when flies are stimulated with UV light and strongly excited in response to a broad range of longer wavelength (VIS) stimuli. Inhibition to UV light is mediated by histaminergic input from R7 and expression of the histamine receptor ort in Dm8, as previously suggested. However, two additional excitatory inputs antagonize the R7 input. First, activation of R8 leads to excitation of Dm8 by non-canonical photoreceptor signaling and cholinergic neurotransmission in the visual circuitry. Second, activation of outer photoreceptors R1-R6 with broad spectral sensitivity causes excitation in Dm8 through the cholinergic medulla interneuron Mi1, which is known for its major contribution to the detection of spatial luminance contrast and visual motion. In summary, Dm8 mediates a second step in UV/VIS color opponent processing in Drosophila by integrating input from all types of photoreceptors. These results demonstrate novel insights into the circuit integration of R1-R6 into color opponent processing and reveal that chromatic and achromatic circuitries of the fly visual system interact more extensively than previously thought (Pagni, 2021).

ide compound eyes, photoreceptors contract to light changes, sharpening retinal images of the moving world in time. Current methods to measure these so-called photoreceptor microsaccades in living insects are spatially limited and technically challenging. this study present goniometric high-speed deep pseudopupil (GHS-DPP) microscopy to assess how the rhabdomeric insect photoreceptors and their microsaccades are organised across the compound eyes. This method enables non-invasive rhabdomere orientation mapping, whilst their microsaccades can be locally light-activated, revealing the eyes' underlying active sampling motifs. By comparing the microsaccades in wild-type Drosophila's open rhabdom eyes to spam-mutant eyes, reverted to an ancestral fused rhabdom state, and honeybee's fused rhabdom eyes, this study showed how different eye types sample light information. These results show different ways compound eyes initiate the conversion of spatial light patterns in the environment into temporal neural signals and highlight how this active sampling can evolve with insects' visual needs (Kemppainen, 2022a).

To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors (Kemppainen, 2022b).

Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head or their body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye. This study shows that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. When the retina moves, visual receptive fields shift accordingly, and even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, it was found that Drosophila perform binocular, vergence movements of their retinas-which could enhance depth perception-when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution (Fenk, 2022).

Animal locomotion is highly adaptive, displaying a large degree of flexibility, yet how this flexibility arises from the integration of mechanics and neural control remains elusive. For instance, animals require flexible strategies to maintain performance as changes in mass or inertia impact stability. Compensatory strategies to mechanical loading are especially critical for animals that rely on flight for survival. To shed light on the capacity and flexibility of flight neuromechanics to mechanical loading, the performance of fruit flies (Drosophila) was pushed near its limit, and a control theoretic framework was implemented. Flies with added inertia were placed inside a virtual reality arena which permitted free rotation about the vertical (yaw) axis. Adding inertia increased the fly's response time yet had little influence on overall gaze stabilization performance. Flies maintained stability following the addition of inertia by adaptively modulating both visuomotor gain and damping. By contrast, mathematical modelling predicted a significant decrease in gaze stabilization performance. Adding inertia altered saccades, however, flies compensated for the added inertia by increasing saccade torque. Taken together, in response to added inertia flies increase reaction time but maintain flight performance through adaptive neural control. Overall, adding inertia decreases closed-loop flight robustness. This work highlights the flexibility and capacity of motor control in flight (Salem, 2023).

Circadian rhythms in physiology and behaviour have near 24 h periodicities that must adjust to the exact 24 h geophysical cycles on earth to ensure adaptive daily timing. Such adjustment is called entrainment. One major mode of entrainment is via the continuous modulation of circadian period by the prolonged presence of light. Although Drosophila melanogaster is a prominent insect model of chronobiology, there is little evidence for such continuous effects of light in the species. This study demonstrates that prolonged light exposure at specific times of the day shapes the daily timing of activity in flies. It was also established that continuous UV- and blue-blocked light lengthens the circadian period of Drosophila, and evidence is provided that this is produced by the combined action of multiple photoreceptors which, includes the cell-autonomous photoreceptor Cryptochrome. Finally, ramped light cycles were introducted as an entrainment paradigm that produces light entrainment that lacks the large light-driven startle responses typically displayed by flies and requires multiple days for entrainment to shifted cycles. These features are reminiscent of entrainment in mammalian models systems and make possible new experimental approaches to understanding the mechanisms underlying entrainment in the fly (Abhilash, 2023).

This study has shown that R8 photoreceptors split visual perception and circadian photoentrainment by co-transmitting two neurotransmitters. This visual segregation is further supported by postsynaptic circuitry in the medulla, such that each unicolumnar neuron mainly receives histaminergic inputs from a single R8 photoreceptor (thus transmitting the fine retinotopic signal), whereas each multicolumnar accessory medulla-innervating, multicolumnar and arcuate neuron (AMA neuron) integrates cholinergic inputs from up to 100 R8 photoreceptor cells (thus integrating the irradiance visual signal). These AMA neurons directly excite downstream clock neurons, forming a shallow three-node circuit for circadian photoentrainment. Thus, this clock-entrainment circuit integrates irradiance signals directly from conventional photoreceptors, bypassing the downstream image-forming processing circuit to avoid a less efficient reconstruction of irradiance signals from the already highly processed signals in the contrast-encoding visual pathways. Notably, the AMA neurons overlap with recently discovered xCEOs that sustain circadian timekeeping of free-running circadian clocks (Tang, 2022). Together, these findings reveal that the AMA neurons and xCEOs play crucial roles in circadian timekeeping, resetting circadian clocks to synchronize their endogenous rhythms to local time by integrating irradiance signals from retinal photoreceptors under LD cycles and sustaining free-running circadian timekeeping through their intrinsic rhythmic electrical oscillations under constant darkness (Xiao, 2023).

The observation that the compound eye-driven electrical responses in clock neurons were reduced by half in the absence of histamine receptors suggests that conventional photoreceptors other than R8 photoreceptors (for example, R1-R6) can use histamine-mediated circuitry pathways to excite clock neurons for circadian photoentrainment. The downstream circuits of R1-R6 photoreceptors might reconstruct irradiance signals from image-forming signals via yet-to-be-uncovered mechanisms as mammalian rod and cone pathways do, indicating the mechanisms underlying irradiance encoding for circadian photoentrainment by conventional photoreceptor pathways might have evolved convergently (Xiao, 2023).

This study also identified an unexpected crosstalk between the image-forming vision and circadian photoentrainment. The chloride channel HisCl1 mediates negative feedback of histamine in R8 photoreceptor cells, thus dynamically reducing photoreceptor depolarization during long light stimulation. This feedback regulation tunes ACh release to avoid its local depletion so that the irradiance signal can be continuously transmitted from R8 photoreceptors to clock neurons during the entire light phase (Xiao, 2023).

This work demonstrates that visual perception and circadian photoentrainment can be segregated as early as the first-order synapses in the visual system, providing a simple yet robust mechanism to enact distinct sensory functions. Furthermore, although cotransmission or co-release of neurotransmitters is an emerging principle in brain research, its behavioural significance remains largely unknown. These finding that cotransmission from the same photoreceptor cells enables segregation and translation of distinct visual features into different behaviours also paves the way for understanding this key, indispensable aspect of the nervous system (Xiao, 2023).

Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function. Conserved orthologs of these are present in most or all eukaryotes including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. Using these criteria, this study molecularly mapped and evaluated the genome sequences of four eye color mutations: chocolate, maroon, mahogany, and red Malpighian tubules. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and in organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome related organelles. Mutants of these genes can provide valuable in vivo models to understand these processes (Grant, 2016).

Patterning and growth are linked during early development and have to be tightly controlled to result in a functional tissue or organ. During the development of the Drosophila eye, this linkage is particularly clear: the growth of the eye primordium mainly results from proliferating cells ahead of the morphogenetic furrow (MF), a moving signaling wave that sweeps across the tissue from the posterior to the anterior side, that induces proliferating cells anterior to it to differentiate and become cell cycle quiescent in its wake. Therefore, final eye disc size depends on the proliferation rate of undifferentiated cells and on the speed with which the MF sweeps across the eye disc. A spatio-temporal model of the growing eye disc was developed in this study based on the regulatory interactions controlled by the signals Decapentaplegic (Dpp), Hedgehog (Hh) and the transcription factor Homothorax (Hth) and how the signaling patterns affect the movement of the MF and impact on eye disc growth was explored. Published and new quantitative data were used to parameterize the model. In particular, two crucial parameter values, the degradation rate of Hth and the diffusion coefficient of Hh, were measured. The model is able to reproduce the linear movement of the MF and the termination of growth of the primordium. It was further shown that the model can explain several mutant phenotypes, but fails to reproduce the previously observed scaling of the Dpp gradient in the anterior compartment (Fried, 2016).

The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-β signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, this study analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer (Rass, 2022).

Negative regulators of the TGF-β signalling pathway are inhibitory Smads (I-Smads), Smurfs and the Ski/Sno protein family. Proteins of the latter group possess two structural domains: the Ski/Sno homology domain and the SMAD4-binding domain. With the help of these domains, Ski/Sno proteins can interact, among others, with R-Smads, N-CoR, Sin3a, SMAD4 and the histone deacetylase HDAC1 and this complex leads to transcriptional repression of target genes. By their expression domains, Ski/Sno proteins can be further subdivided into ubiquitously expressed genes (human Ski and Sno), and mainly neuronally expressed genes, the SKI Family Transcriptional Corepressors (Skor1 and Skor2). The Ski/Sno proteins fulfil a wide range of different physiological functions such as axonal morphogenesis, Purkinje cell development, myogenesis and mammary gland alveogenesis (Rass, 2022).

However, the Ski/Sno proteins were not discovered by their physiological functions but via the transforming capability of the viral ski (v-ski) homologue found in the Sloan-Kettering virus. The first evidence that Ski/Sno proteins possess oncogenic capabilities came from overexpression experiments, where it was shown that the truncation of v-ski is not responsible for the transformation of chicken embryo fibroblasts, but that overexpression of v-ski, Ski or Sno is sufficient for this transformation. Despite this background, their role in carcinogenesis is still not fully understood, if not even contradictory at times. Ski and Sno have been found to be upregulated in different types of cancer e.g. oesophagus squamous cell carcinoma, melanoma, and colorectal cancer. Further evidence for a pro-oncogenic role was found in downregulation analyses of Sno or Ski. This downregulation resulted in decreased tumour growth in breast cancer cells and pancreatic cancer cells. But as stated before, there is some objection that Ski and Sno function purely as oncogenes. Mice, which were heterozygous mutant for Ski or Sno, showed an increased level of tumour induction after carcinogen treatment. In metastatic non-small cell lung cancer, Ski expression is significantly reduced, whereas increased expression of Ski in these cells reduced the invasiveness inhibiting epithelial-mesenchymal transition. Therefore, this could reflect that the outcome of Ski or Sno expression in cancer cells is dependent on the cell type or the actual status of the cancer cells and cancer cells often exploit Ski or Sno to inhibit the anti-proliferative effects of TGF-β signalling. Whereas Ski or Sno have been found to be involved in a lot of different cancer types, there is sparse evidence for deregulation of Skor proteins in cancer cells. Endogenously, Skor proteins have been linked to neurodevelopmental processes. After Skor1 overexpression, genes involved in axonal guidance or post-synapse assembly were differentially expressed. Skor2 is important for cerebellar Purkinje cell differentiation as in Skor2 knockout mice dendrite formation of Purkinje cells was impaired. Pathophysiologically, Skor1 has mainly been linked to restless leg syndrome and localized scleroderma (Rass, 2022).

In Drosophila melanogaster, only one homologue of Ski and Sno, which is designated Snoo, and one homologue of Skor1 and Skor2, which is designated Fuss, exist. It has been recently shown that Fuss is interacting with SMAD4 and HDAC1. In overexpression assays, Fuss can inhibit Dpp signalling and endogenously, the Fuss/HDAC1 complex is required for bitter gustatory neuron differentiation and fuss mutant flies pause more often during walking. However, this study was interested whether the Skor/Fuss proteins retained their ability to inhibit differentiation and induce increased proliferation. For this purpose, Fuss was overexpressed in differentiating cells of the eye imaginal disc, an excellent model tissue to study regulatory gene function in the context of carcinogenesis. This overexpression impaired photoreceptor axon guidance and inhibited the differentiation of accessory cells such as cone cells and primary pigment cells, which are all transformed into a basal pigment cell type. In a second approach, fuss overexpressing clones were generated early during development in the eye imaginal discs, when cells are still proliferating. This resulted in vast outgrowths of undifferentiated tissue of the eye imaginal disc because fuss overexpression most likely inhibited Dpp-signalling, a member of the TGF-β superfamily. This work shows that Fuss retained the ability of Ski/Sno proteins to inhibit the antiproliferative effects of TGF-β signalling by analogous inhibition of Dpp-signalling, allowing proliferation to be sustained (Rass, 2022).

The overexpression of fuss posterior to the morphogenetic furrow with the GMR-Gal4 driver line resulted in a nearly complete loss of all cell types in the adult eye. During development, photoreceptor axons were not able to target the appropriate layers of the optic lobe anymore and cone cells, primary pigment cells and bristle cells were transformed into a basal pigment cell fate. This transformation was caused by the inhibition of sv expression, which is crucial for accessory cell differentiation. Additionally, increased apoptosis during pupal development lead to the removal of photoreceptors and lastly adult eyes only consisted of cells containing pigment granules. This lack of differentiation cannot be explained by the Dpp inhibiting role Fuss exerts, when overexpressed, because inhibiting the Dpp signaling pathway via knockdown of Tkv or Med had no effect. Photoreceptor axon guidance is impaired, if Dpp signaling is disrupted in photoreceptors by the expression of the inhibitory Smad Dad. Thus, the observed photoreceptor axon guidance phenotype, when fuss is overexpressed with GMR, could indeed be a result of Dpp signaling inhibition. However, the loss of nearly all eye cell types is due to other effects (e.g. downregulation of sv and apoptosis) than Dpp signaling repression alone, because loss of Dpp signaling behind the morphogenetic furrow only results in mild patterning defects of the pupal retina. Nonetheless, the inhibition of cell differentiation has already been shown in other cancer models e.g., when two copies of the constitutive active form of the receptor tyrosine kinase dRETMEN2B are expressed with the GMR-Gal4 line, pupal retinas are devoid of any distinguishable cell types. This phenotype is indistinguishable from the phenotype of the pupal retinas generated by the overexpression of fuss via GMR-Gal4. In a screen for novel oncogenes from breast cancer patients, human transgenes have been overexpressed with the GMR-Gal4 driver line. Overexpression of human RPS12, a subunit of the small ribosomal subunit, whose expression is increased in various cancer types, leads also to a glazed eye phenotype. Therefore, different oncogenes can result in different outcomes when expressed with the GMR-Gal4 driver line and are not always leading to massive tissue overgrowth like the Yorkie overexpression. Most importantly, with this approach to overexpress fuss in cells which already were destined for acquiring a cell fate and have left the cell cycle, it was not possible to induce increased proliferation anymore, but could prevent cell differentiation (Rass, 2022).

Consequently, a more pluripotent cell type in the eye imaginal disc was used and fuss overexpressing clones were induced prior to the formation of the morphogenetic furrow. These results allowed the assumption that in this context, fuss overexpressing clones do not react to the antiproliferative effects of the Dpp morphogen anymore. Instead, wg expression and thus, proliferation promotion might be maintained. This leads to outgrowths of clonal tissue from the eye imaginal disc of third instar larvae, which showed an increased number of mitotic events. If these flies survived to adulthood, undifferentiated, extra tissue was visible in the complex eye (Rass, 2022).

An analogous mechanism can be observed in tumors which overexpress Ski or Sno. The TGF-β signaling pathway is also anti-proliferative, but this action is inhibited by the increased presence of Ski/Sno proteins. Therefore, the molecular mode of action is similar to the human Ski/Sno proteins. The function of Ski and Sno is highly context dependent, as they can fulfill an anti-oncogenic or pro-oncogenic role depending on the cancer type or status of the cancer. This was also observed with fuss overexpressing clones. Only when induced 48h after egg laying, was additional tissue found in late third instar larvae and only in eye imaginal discs, because there, Dpp counteracts the proliferative effects of Wg signaling. When fuss is overexpressed in the wing disc or after induction of the morphogenetic furrow differentiation is inhibited, this results in a wing with truncated veins or in a smooth eye surface. This is also underlined by RNAseq data from eye and wing imaginal discs, where fuss was overexpressed with the GMR-Gal4 and Nub-Gal4 driver line, respectively. In the eye dataset, wg expression in eye imaginal discs is not significantly different from control eye discs, whereas wg expression in fuss overexpression wing discs is significantly reduced in contrast to control wing discs (Rass, 2022).

Thus, this study showed that the Skor protein Fuss in Drosophila melanogaster still retained the function of the Ski/Sno proteins by inhibiting differentiation but inducing hyperproliferation. But the hallmarks of real tumorigenesis are lacking, because at some point during pupal development, proliferation stops, and these cells become protruding head tissue as it could be observed in complex eyes of surviving flies. Furthermore, there was no evidence of an epithelial-mesenchymal transition because fuss overexpressing clones maintained their epithelial fate. It will be of high interest if future studies can find similar results in overexpression studies for the vertebrate Skor proteins or detect increased expression of these proteins in specific cancer types (Rass, 2022).

The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, this study used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, in situ hybridization was conducted at 50% pupal development, a key age for lens secretion. The results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes (Stahl, 2017).

This study demonstrates that autophagy, an evolutionarily conserved self-degradation process of eukaryotic cells, is essential for eye development in Drosophila. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. The results also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by labial. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought (Billes, 2018).