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Connections between neurons can be mapped by acquiring and analysing electron microscopic brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative 1-6 , but nevertheless inadequate for understanding brain function more globally. Here we present a neuronal wiring diagram of a whole brain containing 5 × 10 7 chemical synapses 7 between 139,255 neurons reconstructed from an adult female Drosophila melanogaster 8,9 . The resource also incorporates annotations of cell classes and types, nerves, hemilineages and predictions of neurotransmitter identities 10-12 . Data products are available for download, programmatic access and interactive browsing and have been made interoperable with other fly data resources. We derive a projectome-a map of projections between regions-from the connectome and report on tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine and descending neurons) across both hemispheres and between the central brain and the optic lobes. Tracing from a subset of photoreceptors to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviours. The technologies and open ecosystem reported here set the stage for future large-scale connectome projects in other species., (© 2024. The Author(s).)

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells., (© 2024. The Author(s).)

Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the progression across the tissue of an epithelial fold known as the morphogenetic furrow. Morphogenetic furrow progression involves apical cell constriction and movement of apical cell edges. Here, we show that cells progressing through the morphogenetic furrow move their basal edges in opposite direction to their apical edges, resulting in a cellular tilting movement. We further demonstrate that cells generate, at their basal side, oriented, force-generating protrusions. Knockdown of the protein kinase Src42A or photoactivation of a dominant-negative form of the small GTPase Rac1 reduces protrusion formation. Impaired protrusion formation stalls basal cell movement and slows down morphogenetic furrow progression and photoreceptor differentiation. This work identifies a cellular tilting mechanism important for the generation of dynamic tissue shape changes and cell differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A complex network of transcription factor interactions propagates across the larval eye disc to establish columns of evenly-spaced R8 precursor cells, the founding cells of Drosophila ommatidia. After the recruitment of additional photoreceptors to each ommatidium, the surrounding cells are organized into their stereotypical pattern during pupal development. These support cells - comprised of pigment and cone cells - are patterned to encapsulate the photoreceptors and separate ommatidia with an hexagonal honeycomb lattice. Since the proteins and processes essential for correct eye patterning are conserved, elucidating how these function and change during Drosophila eye patterning can substantially advance our understanding of transcription factor and signaling networks, cytoskeletal structures, adhesion complexes, and the biophysical properties of complex tissues during their morphogenesis. Our understanding of many of these aspects of Drosophila eye patterning is largely descriptive. Many important questions, especially relating to the regulation and integration of cellular events, remain., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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. The underlying cis-regulatory logic remains poorly understood, but 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. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find 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. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins., Competing Interests: The authors have declared that no competing interests exist.

Diversification of neuronal subtypes often requires stochastic gene regulatory mechanisms. How stochastically expressed transcription factors interact with other regulators in gene networks to specify cell fates is poorly understood. The random mosaic of color-detecting R7 photoreceptor subtypes in Drosophila is controlled by the stochastic on/off expression of the transcription factor Spineless (Ss). In Ss ON R7s, Ss induces expression of Rhodopsin 4 (Rh4), whereas in Ss OFF R7s, the absence of Ss allows expression of Rhodopsin 3 (Rh3). Here, we find that the transcription factor Runt, which is initially expressed in all R7s, is sufficient to promote stochastic Ss expression. Later, as R7s develop, Ss negatively feeds back onto Runt to prevent repression of Rh4 and ensure proper fate specification. Together, stereotyped and stochastic regulatory inputs are integrated into feedforward and feedback mechanisms to control cell fate., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

As an obligate ectoparasite of bats, the bat fly Trichobius frequens (Diptera: Streblidae) inhabits the same subterranean environment as their nocturnal bat hosts. In this study, we characterize the macromorphology, optical architecture, rhabdom anatomy, photoreceptor absorbance, and opsin expression of the significantly reduced visual system in T. frequens resulting from evolution in the dark. The eyes develop over a 21-22 day pupal developmental period, with pigmentation appearing on pupal day 11. After eclosion as an adult, T. frequens eyes consist of on average 8 facets, each overlying a fused rhabdom consisting of anywhere from 11 to 18 estimated retinula cells. The dimensions of the facets and fused rhabdoms are similar to those measured in other nocturnal insects. T. frequens eyes are functional as shown by expression of a Rh1 opsin forming a visual pigment with a peak sensitivity to 487 nm, similar to other dipteran Rh1 opsins. Future studies will evaluate how individuals with such reduced capabilities for spatial vision as well as sensitivity still capture enough visual information to use flight to maneuver through dark habitats., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we 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 our understanding of apical growth during morphogenesis., (© 2020 Hebbar et al.)

Ongoing research using cell transplantation and viral-mediated gene therapy has been making progress to restore vision by retinal repair, but targeted delivery and complete cellular integration remain challenging. An alternative approach is to induce endogenous Müller glia (MG) to regenerate lost neurons and photoreceptors, as occurs spontaneously in teleost fish and amphibians. Extracellular vesicles (EVs) can transfer protein and RNA cargo between cells serving as a novel means of cell-cell communication. We conducted an in vivo screen in zebrafish to identify sources of EVs that could induce MG to dedifferentiate and generate proliferating progenitor cells after intravitreal injection into otherwise undamaged zebrafish eyes. Small EVs (sEVs) from C6 glioma cells were the most consistent at inducing MG-derived proliferating cells. Ascl1a expression increased after intravitreal injection of C6 sEVs and knockdown of ascl1a inhibited the induction of proliferation. Proteomic and RNAseq analyses of EV cargo content were performed to begin to identify key factors that might target EVs to MG and initiate retina regeneration., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. We demonstrate that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells., Competing Interests: The authors have declared that no competing interests exist.

Morphologically diverse eyes have evolved numerous times, yet little is known about how eye gain and loss is related to photic environment. The pteriomorphian bivalves (e.g., oysters, scallops, and ark clams), with a remarkable range of photoreceptor organs and ecologies, are a suitable system to investigate the association between eye evolution and ecological shifts. The present phylogenetic framework was based on amino acid sequences from transcriptome datasets and nucleotide sequences of five additional genes. In total, 197 species comprising 22 families from all five pteriomorphian orders were examined, representing the greatest taxonomic sampling to date. Morphological data were acquired for 162 species and lifestyles were compiled from the literature for all 197 species. Photoreceptor organs occur in 11 families and have arisen exclusively in epifaunal lineages, that is, living above the substrate, at least five times independently. Models for trait evolution consistently recovered higher rates of loss over gain. Transitions to crevice-dwelling habit appear associated with convergent gains of eyespots in epifaunal lineages. Once photoreceptor organs have arisen, multiple losses occurred in lineages that shift to burrowing lifestyles and deep-sea habitats. The observed patterns suggest that eye evolution in pteriomorphians might have evolved in association with light-guided behaviors, such as phototaxis, body posture, and alarm responses., (© 2020 The Authors. Evolution © 2020 The Society for the Study of Evolution.)

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep-wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ., (© 2019 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina., Competing Interests: The authors have declared that no competing interests exist.

The establishment of the functional nervous system requires coordinated development of neurons and glia in the embryo. Our understanding of underlying molecular and cellular mechanisms, however, remains limited. The developing Drosophila visual system is an excellent model for understanding the developmental control of the nervous system. By performing a systematic transgenic RNAi screen, we investigated the requirements of secreted proteins and cell-surface receptors for the development of photoreceptor neurons (R cells) and wrapping glia (WG) in the Drosophila visual system. From the screen, we identified seven genes whose knockdown disrupted the development of R cells and/or WG, including amalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific analysis revealed that ama is required in the developing eye disc for promoting cell proliferation and differentiation, which is essential for the migration of glia in the optic stalk. Our results also suggest that nrg functions in both eye disc and WG for coordinating R-cell and WG development.

Drosophila melanogaster photoreceptor cells are highly polarized epithelial cells. Their apical membrane is further subdivided into the stalk membrane and the light-sensing rhabdomere. The photo-pigment Rhodopsin1 (Rh1) localizes to the rhabdomere, whereas the apical determinant Crumbs (Crb) is enriched at the stalk membrane. The proteoglycan Eyes shut (Eys) is secreted through the apical membrane into an inter-rhabdomeral space. Rh1, Crb, and Eys are essential for the development of photoreceptor cells, normal vision, and photoreceptor cell survival. Human orthologs of all three proteins have been linked to retinal degenerative diseases. Here, we describe an RNAi-based screen examining the importance of 237 trafficking-related genes in apical trafficking of Eys, Rh1, and Crb. We found 28 genes that have an effect on the localization and/or levels of these apical proteins and analyzed several factors in more detail. We show that the Arf GEF protein Sec71 is required for biosynthetic traffic of both apical and basolateral proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1, and that Syntaxin 7/Avalanche controls the endocytosis of Rh1, Eys, and Crb., (Copyright © 2019 Laffafian, Tepass.)

Drosophila R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11's binding partner DIP-γ, while pR7 PRs synapse onto DIP-γ-negative pDm8. Dpr11 and DIP-γ expression patterns define 'yellow' and 'pale' color vision circuits. We examined Dm8 neurons in these circuits by electron microscopic reconstruction and expansion microscopy. DIP-γ and dpr11 mutations affect the morphologies of yDm8 distal ('home column') dendrites. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 PRs, and interactions between Dpr11 and DIP-γ are required for yDm8 survival. These interactions also allow yDm8 neurons to select yR7 PRs as their appropriate home column partners. yDm8 and pDm8 neurons do not normally compete for survival signals or R7 partners, but can be forced to do so by manipulation of R7 subtype fate., Competing Interests: KM, VK, ST, MA, KZ No competing interests declared, (© 2019, Menon et al.)

Sensory systems use stochastic fate specification to increase their repertoire of neuronal types. How these stochastic decisions are coordinated with the development of their targets is unknown. In the Drosophila retina, two subtypes of ultraviolet-sensitive R7 photoreceptors are stochastically specified. In contrast, their targets in the brain are specified through a deterministic program. We identified subtypes of the main target of R7, the Dm8 neurons, each specific to the different subtypes of R7s. Dm8 subtypes are produced in excess by distinct neuronal progenitors, independently from R7. After matching with their cognate R7, supernumerary Dm8s are eliminated by apoptosis. Two interacting cell adhesion molecules, Dpr11 and DIPγ, are essential for the matching of one of the synaptic pairs. These mechanisms allow the qualitative and quantitative matching of R7 and Dm8 and thereby permit the stochastic choice made in R7 to propagate to the brain., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We exploited the high Ras activity induced differentiation of supernumerary R7 cells in Drosophila eyes to examine if hsrω lncRNAs influence active Ras signaling. Surprisingly, either down- or up-regulation of hsrω lncRNAs in sev-GAL4>Ras V12 expressing eye discs resulted in complete pupal lethality and substantially greater increase in R7 photoreceptor number at the expense of cone cells. Enhanced nuclear p-MAPK and presence of sev-GAL4 driven Ras V12 bound RafRBDFLAG in cells not expressing the sev-GAL4 driver indicated non-cell autonomous spread of Ras signaling when hsrω levels were co-altered. RNA-sequencing revealed that down-and up-regulation of hsrω transcripts in sev-GAL4>Ras V12 expressing eye discs elevated transcripts of positive or negative modulators, respectively, of Ras signaling so that either condition enhances it. Altered hsrω transcript levels in sev-GAL4>Ras V12 expressing discs also affected sn/sno/sca RNAs and some other RNA processing transcript levels. Post-transcriptional changes due to the disrupted intra-cellular dynamicity of omega speckle associated hnRNPs and other RNA-binding proteins that follow down- or up-regulation of hsrω lncRNAs appear to be responsible for the further elevated Ras signaling. Cell autonomous and non-autonomous enhancement of Ras signaling by lncRNAs like hsrω has implications for cell signaling during high Ras activity commonly associated with some cancers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Drosophila photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, we found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein syndapin is essential for apical membrane segregation. The analysis of the super-resolution microscopy, STORM and the electron microscopy suggest that syndapin localizes to the neck of the microvilli at the base of the rhabdomere. Syndapin and moesin are required to constrict the neck of the microvilli to organize the membrane architecture at the base of the rhabdomere, to exclude the stalk membrane. Simultaneous loss of syndapin along with the microvilli adhesion molecule chaoptin significantly enhanced the disruption of stalk-rhabdomere segregation. However, loss of the factors involving endocytosis do not interfere. These results indicated syndapin is most likely functioning through its membrane curvature properties, and not through endocytic processes for stalk-rhabdomere segregation. Elucidation of the mechanism of this unconventional domain formation will provide novel insights into the field of cell biology., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Many insects have triplets of camera type eyes, called ocelli, whose function remains unclear for most species. Here, we investigate the ocelli of the bumblebee, Bombus terrestris, using reconstructed 3D data from X-ray microtomography scans combined with computational ray-tracing simulations. This method enables us, not only to predict the visual fields of the ocelli, but to explore for the first time the effect that hair has on them as well as the difference between worker female and male ocelli. We find that bumblebee ocellar fields of view are directed forward and dorsally, incorporating the horizon as well as the sky. There is substantial binocular overlap between the median and lateral ocelli, but no overlap between the two lateral ocelli. Hairs in both workers and males occlude the ocellar field of view, mostly laterally in the worker median ocellus and dorsally in the lateral ocelli. There is little to no sexual dimorphism in the ocellar visual field, suggesting that in B. terrestris they confer no advantage to mating strategies. We compare our results with published observations for the visual fields of compound eyes in the same species as well as with the ocellar vision of other bee and insect species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Stereotyped synaptic connections define the neural circuits of the brain. In vertebrates, stimulus-independent activity contributes to neural circuit formation. It is unknown whether this type of activity is a general feature of nervous system development. Here, we report patterned, stimulus-independent neural activity in the Drosophila visual system during synaptogenesis. Using in vivo calcium, voltage, and glutamate imaging, we found that all neurons participate in this spontaneous activity, which is characterized by brain-wide periodic active and silent phases. Glia are active in a complementary pattern. Each of the 15 of over 100 specific neuron types in the fly visual system examined exhibited a unique activity signature. The activity of neurons that are synaptic partners in the adult was highly correlated during development. We propose that this cell-type-specific activity coordinates the development of the functional circuitry of the adult brain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The ascidian larva has a pigmented ocellus comprised of a cup-shaped array of approximately 30 photoreceptor cells, a pigment cell, and three lens cells. Morphological, physiological and molecular evidence has suggested evolutionary kinship between the ascidian larval photoreceptors and vertebrate retinal and/or pineal photoreceptors. Rx, an essential factor for vertebrate photoreceptor development, has also been suggested to be involved in the development of the ascidian photoreceptor cells, but a recent revision of the photoreceptor cell lineage raised a crucial discrepancy between the reported expression patterns of Rx and the cell lineage. Here, we report spatio-temporal expression patterns of Rx at single-cell resolution along with mitotic patterns up to the final division of the photoreceptor-lineage cells in Ciona. The expression of Rx commences in non-photoreceptor a-lineage cells on the right side of the anterior sensory vesicle at the early tailbud stage. At the mid tailbud stage, Rx begins to be expressed in the A-lineage photoreceptor cell progenitors located on the right side of the posterior sensory vesicle. Thus, Rx is specifically but not exclusively expressed in the photoreceptor-lineage cells in the ascidian embryo. Two cis-regulatory modules are shown to be important for the photoreceptor-lineage expression of Rx. The cell division patterns of the photoreceptor-lineage cells rationally explain the generation of the cup-shaped structure of the pigmented ocellus. The present findings demonstrate the complete cell lineage of the ocellus photoreceptor cells and provide a framework elucidating the molecular and cellular mechanisms of photoreceptor development in Ciona., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP 2 ) generating diacylglycerol (DAG), inositol trisphosphate and H + . This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

During development, transcriptional complexes at enhancers regulate gene expression in complex spatiotemporal patterns. To achieve robust expression without spurious activation, the affinity and specificity of transcription factor - DNA interactions must be precisely balanced. Protein - protein interactions among transcription factors are also critical, yet how their affinities impact enhancer output is not understood. The Drosophila transcription factor Yan provides a well-suited model to address this, as its function depends on the coordinated activities of two independent and essential domains: the DNA-binding ETS domain and the self-associating SAM domain. To explore how protein - protein affinity influences Yan function, we engineered mutants that increase SAM affinity over four orders of magnitude. This produced a dramatic subcellular redistribution of Yan into punctate structures, reduced repressive output and compromised survival. Cell-type specification and genetic interaction defects suggest distinct requirements for polymerization in different regulatory decisions. We conclude that tuned protein - protein interactions enable the dynamic spectrum of complexes that are required for proper regulation., Competing Interests: CH, JW, ST, IR No competing interests declared, (© 2018, Hope et al.)

Photoreceptor (PR) axons project from the retina to the optic lobe in brain and form a precise retinotopic map in the Drosophila visual system. Yet the role of retinal basal glia in the retinotopic map formation is not previously known. We examined the formation of the retinotopic map by marking single PR pairs and following their axonal projections. In addition to confirming previous studies that the spatial information is preserved from the retina to the optic stalk and then to the optic lamina, we found that the young PR R3/4 axons transiently overshoot and then retract to their final destination, the lamina plexus. We then examined the process of wrapping glia (WG) membrane extension in the eye disc and showed that the WG membrane extensions also follow the retinotopic map. We show that the WG is important for the proper spatial distribution of PR axons in the optic stalk and lamina, suggesting an active role of wrapping glia in the retinotopic map formation.

The palm borer moth Paysandisia archon (Burmeister, 1880) (fam. Castniidae) is a large, diurnally active palm pest. Its compound eyes consist of ~ 20,000 ommatidia and have apposition optics with interommatidial angles below 1°. The ommatidia contain nine photoreceptor cells and appear structurally similar to those in nymphalid butterflies. Two morphological ommatidial types were identified. Using the butterfly numbering scheme, in type I ommatidia, the distal rhabdom consists exclusively of the rhabdomeres of photoreceptors R1-2; the medial rhabdom has contributions from R1-8. The rhabdom in type II ommatidia is distally split into two sub-rhabdoms, with contributions from photoreceptors R2, R3, R5, R6 and R1, R4, R7, R8, respectively; medially, only R3-8 and not R1-2 contribute to the fused rhabdom. In both types, the pigmented bilobed photoreceptors R9 contribute to the rhabdom basally. Their nuclei reside in one of the lobes. Upon light adaptation, in both ommatidial types, the rhabdoms secede from the crystalline cones and pigment granules invade the gap. Intracellular recordings identified four photoreceptor classes with peak sensitivities in the ultraviolet, blue, green and orange wavelength regions (at 360, 465, 550, 580 nm, respectively). We discuss the eye morphology and optics, the photoreceptor spectral sensitivities, and the adaptation to daytime activity from a phylogenetic perspective.

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.

Many visual animals exploit spectral information for seeking food and mates, for identifying preys and predators, and for navigation. Animals use chromatic information in two ways. "True color vision," the ability to discriminate visual stimuli on the basis of their spectral content independent of brightness, is thought to play an important role in object identification. In contrast, "wavelength-specific behavior," which is strongly dependent on brightness, often associates with foraging, navigation, and other species-specific needs. Among animals capable of chromatic vision, insects, with their diverse habitats, stereotyped behaviors, well-characterized anatomy and powerful genetic tools, are attractive systems for studying chromatic information processing. In this review, we first discuss insect photoreceptors and the relationship between their spectral sensitivity and animals' color vision and ecology. Second, we review recent studies that dissect chromatic circuits and explore neural mechanisms of chromatic information processing. Finally, we review insect behaviors involving "true color vision" and "wavelength-specific behaviors," especially in bees, butterflies, and flies. We include examples of high-order color vision, such as color contrast and constancy, which are shared by vertebrates. We focus on Drosophila studies that identified neuronal correlates of color vision and innate spectral preferences. We also discuss the electrophysiological studies in bees that reveal color encoding. Despite structural differences between insects' and vertebrates' visual systems, their chromatic vision appears to employ the same processing principles, such as color opponency, suggesting convergent solutions of neural computation to common problems.

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not in their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologs, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins in establishing visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1 -dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The Drosophila visual system has become a premier model for probing how neural diversity is generated during development. Recent work has provided deeper insight into the elaborate mechanisms that control the range of types and numbers of neurons produced, which neurons survive, and how they interact. These processes drive visual function and influence behavioral preferences. Other studies are beginning to provide insight into how neuronal diversity evolved in insects by adding new cell types and modifying neural circuits. Some of the most powerful comparisons have been those made to the Drosophila visual system, where a deeper understanding of molecular mechanisms allows for the generation of hypotheses about the evolution of neural anatomy and function. The evolution of new neural types contributes additional complexity to the brain and poses intriguing questions about how new neurons interact with existing circuitry. We explore how such individual changes in a variety of species might play a role over evolutionary timescales. Lessons learned from the fly visual system apply to other neural systems, including the fly central brain, where decisions are made and memories are stored.

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. Here, we determine 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 investigate whether cell cycle phase-specific arrest influences the fate of these cells. We demonstrate that retinal precursor cells that arrest in G2 during larval development are 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. We conclude that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. Our 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Asymmetries are observed in a great number of taxa in metazoans. More particularly, functional lateralization and neuroanatomical asymmetries within the central nervous system have been a matter of intense research for at least two hundred years. While asymmetries of some paired structures/organs (e.g. eyes, ears, kidneys, legs, arms) constitute random deviations from a pure bilateral symmetry, brain asymmetries such as those observed in the cortex and epithalamus are directional. This means that molecular and anatomical features located on one side of a given structure are observed in most individuals. For instance, in humans, the neuronal tract connecting the language areas is enlarged in the left hemisphere. When asymmetries are fixed, their molecular mechanisms can be studied using mutants displaying different phenotypes: left or right isomerism of the structure, reversed asymmetry or random asymmetry. Our understanding of asymmetry in the nervous system has been widely enriched thanks to the characterization of mutants affecting epithalamus asymmetry. Furthermore, two decades ago, pioneering studies revealed that a specific morphogen, Nodal, active only on one side of the embryo during development is an important molecule in asymmetry patterning. In this review, I have gathered important data bringing insight into the origin and evolution of epithalamus asymmetry and the role of Nodal in metazoans. After a short introduction on brain asymmetries (chapter I), I secondly focus on the molecular and anatomical characteristics of the epithalamus in vertebrates and explore some functional aspects such as its photosensitive ability related to the pineal complex (chapter II). Third, I discuss homology relationship of the parapineal organ among vertebrates (chapter III). Fourth, I discuss the possible origin of the epithalamus, presenting cells displaying photosensitive properties and/or asymmetry in the anterior part of the body in non-vertebrates (chapter IV). Finally, I report Nodal signaling expression data and functional experiments performed in different metazoan groups (chapter V)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuronal birth and specification must be coordinated across the developing brain to generate the neurons that constitute neural circuits. We used the Drosophila visual system to investigate how development is coordinated to establish retinotopy, a feature of all visual systems. Photoreceptors achieve retinotopy by inducing their target field in the optic lobe, the lamina neurons, with a secreted differentiation cue, epidermal growth factor (EGF). We find that communication between photoreceptors and lamina cells requires a signaling relay through glia. In response to photoreceptor-EGF, glia produce insulin-like peptides, which induce lamina neuronal differentiation. Our study identifies a role for glia in coordinating neuronal development across distinct brain regions, thus reconciling the timing of column assembly with that of delayed differentiation, as well as the spatiotemporal pattern of lamina neuron differentiation., (Copyright © 2017, American Association for the Advancement of Science.)

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca 2+ influx in response to light, but whether Ca 2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca 2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% F max with ∼1200 effectively absorbed photons and saturated (ΔF/F 0 ∼10-20) with 10000-30000 photons. In Ca 2+ free bath, smaller (ΔF/F 0 ∼4), long latency (∼200ms) light-induced Ca 2+ rises were still detectable. These were unaffected in InsP 3 receptor mutants, but virtually eliminated when Na + was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca 2+ free rises were also eliminated in Na + /Ca 2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca 2+ free rises are strictly dependent on Na + influx and activity of the exchanger, suggesting they reflect re-equilibration of Na + /Ca 2+ exchange across plasma or intracellular membranes following massive Na + influx. Any tiny Ca 2+ free rise remaining without exchanger activity was equivalent to <10nM (ΔF/F 0 ∼0.1), and unlikely to play any role in phototransduction., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Flying is often associated with superior visual performance, as good vision is crucial for detection and implementation of rapid visually guided aerial movements. To understand the evolution of insect visual systems it is therefore important to compare phylogenetically related species with different investments in flight capability. Here, we describe and compare morphological and electrophysiological properties of photoreceptors from the habitually flying green cockroach Panchlora nivea and the American cockroach Periplaneta americana , which flies only at high ambient temperatures. In contrast to Periplaneta , ommatidia in Panchlora were characterized by two-tiered rhabdom, which might facilitate detection of polarized light while flying in the dark. In patch-clamp experiments, we assessed the absolute sensitivity to light, elementary and macroscopic light-activated current and voltage responses, voltage-activated potassium (K v ) conductances, and information transfer. Both species are nocturnal, and their photoreceptors were similarly sensitive to light. However, a number of important differences were found, including the presence in Panchlora of a prominent transient K v current and a generally low variability in photoreceptor properties. The maximal information rate in Panchlora was one-third higher than in Periplaneta , owing to a substantially higher gain and membrane corner frequency. The differences in performance could not be completely explained by dissimilarities in the light-activated or K v conductances; instead, we suggest that the superior performance of Panchlora photoreceptors mainly originates from better synchronization of elementary responses. These findings raise the issue of whether the evolutionary tuning of photoreceptor properties to visual demands proceeded differently in Blattodea than in Diptera., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates.

Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase ε 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.

The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates. Photoreceptor cells of the pigmented ocellus have been thought to develop from anterior animal (a-lineage) blastomeres, whereas the developmental origin of the nonpigmented ocellus has not been determined. Here, we show that the photoreceptor cells of both ocelli develop from the right anterior vegetal hemisphere: those of the pigmented ocellus from the right A9.14 cell and those of the nonpigmented ocellus from the right A9.16 cell. The pigmented ocellus is formed by a combination of two lineages of cells with distinct embryonic origins: the photoreceptor cells originate from a medial portion of the A-lineage neural plate, while the pigment cell originates from the lateral edge of the a-lineage neural plate. In light of the recently proposed close evolutionary relationship between the ocellus pigment cell of ascidians and the cephalic neural crest of vertebrates, the ascidian ocellus may represent a prototypic contribution of the neural crest to a cranial sensory organ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A quick guide to the diverse and unusual eyes of polychaete fan worms, by Michael Bok and Dan-Eric Nilsson., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, our findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults., (© 2016 Federation of European Biochemical Societies.)

The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to 'cis'-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader applicability than has been previously recognized.

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected. The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.

Visually guided flight control in the rainforest is arguably one of the most complex insect behaviors: illumination varies dramatically depending on location [1], and the densely cluttered environment blocks out most of the sky [2]. What visual information do insects sample for flight control in this habitat? To begin answering this question, we determined the visual fields of the ocelli-thought to play a role in attitude stabilization of some flying insects [3-5]-of an orchid bee, Euglossa imperialis. High-resolution 3D models of the ocellar system from X-ray microtomography were used for optical ray tracing simulations. Surprisingly, these showed that each ocellus possesses two distinct visual fields-a focused monocular visual field suitable for detecting features elevated above the horizon and therefore assisting with flight stabilization [3-5] and, unlike other ocelli investigated to date [4, 6, 7], a large trinocular fronto-dorsal visual field shared by all ocelli. Histological analyses show that photoreceptors have similar orientations within each ocellus and are likely to be sensitive to polarized light, as in some other hymenopterans [7, 8]. We also found that the average receptor orientation is offset between the ocelli, each having different axes of polarization sensitivity relative to the head. Unlike the eyes of any other insect described to date, this ocellar system meets the requirements of a true polarization analyzer [9, 10]. The ocelli of E. imperialis could provide sensitive compass information for navigation in the rainforest and, additionally, provide cues for visual discrimination or flight control., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Eye development requires an evolutionarily conserved group of transcription factors, termed the retinal determination network (RDN). However, little is known about the molecular mechanism by which the RDN instructs cells to differentiate into photoreceptors. We show that photoreceptor cell identity in Drosophila is critically regulated by the transcription factor Glass, which is primarily expressed in photoreceptors and whose role in this process was previously unknown. Glass is both required and sufficient for the expression of phototransduction proteins. Our results demonstrate that the RDN member Sine oculis directly activates glass expression, and that Glass activates the expression of the transcription factors Hazy and Otd. We identified hazy as a direct target of Glass. Induced expression of Hazy in the retina partially rescues the glass mutant phenotype. Together, our results provide a transcriptional link between eye field specification and photoreceptor differentiation in Drosophila, placing Glass at a central position in this developmental process., (© 2016. Published by The Company of Biologists Ltd.)

Ants are unusual among insects in that individuals of the same species within a single colony have different modes of locomotion and tasks. We know from walking ants that vision plays a significant role in guiding this behaviour, but we know surprisingly little about the potential contribution of visual sensory structures for a flying mode of locomotion. Here we investigate the structure of the compound eye and ocelli in pedestrian workers, alate females and alate males of an Australian ant, Camponotus consobrinus, and discuss the trade-offs involved in optical sensitivity and spatial resolution. Male ants have more but smaller ommatidia and the smallest interommatidial angles, which is most likely an adaptation to visually track individual flying females. Both walking and flying forms of ants have a similar proportion of specialized receptors sensitive to polarized skylight, but the absolute number of these receptors varies, being greatest in males. Ocelli are present only in the flying forms. Each ocellus consists of a bipartite retina with a horizon-facing dorsal retina, which contains retinula cells with long rhabdoms, and a sky-facing ventral retina with shorter rhabdoms. We discuss the implications of these and their potential for sensing the pattern of polarized skylight.

The origin of the Drosophila compound eye predates the ancestor of Pancrustacea, the arthropod clade that includes insects and Crustaceans. Recent studies in emerging model systems for pancrustacean development-the red flour beetle Tribolium castaneum and water flea Daphnia pulex-have begun to shed light on the evolutionary conservation of transcriptional mechanisms found for the Drosophila compound eye. Here, we discuss the conserved roles of the transcription factors Otd and Pph13, which complement each other in two terminal events of photoreceptor differentiation: rhabdomere morphogenesis and transcriptional default activation of opsin gene expression. The synthesis of these data allows us to frame an evolutionary developmental model of the earliest events that generated the wavelength-specific photoreceptor subtypes of pancrustacean compound eyes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, Tb(R332H) (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca(2+)-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca(2+)- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle., (© 2015. Published by The Company of Biologists Ltd.)