A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation.

Drosophila rhabdomeric terminal photoreceptor differentiation is an extended process taking several days to complete. Following ommatidial patterning by the morphogenetic furrow, photoreceptors are sequentially recruited and specified, and terminal differentiation begins. Key events of terminal differentiation include the establishment of apical and basolateral domains, rhabdomere and stalk formation, inter-rhabdomeral space formation, and expression of phototransduction machinery. While many key regulators of these processes have been identified, the complete network of transcription factors to downstream effector molecules necessary for regulating each of these major events remains incomplete. Here, we report an RNAi screen to identify additional molecules and cellular pathways required for photoreceptor terminal differentiation. First, we tested several eye-specific GAL4 drivers for correct spatial and temporal specificity and identified Pph13-GAL4 as the most appropriate GAL4 line for our screen. We screened lines available through the Transgenic RNAi Project and isolated lines that when combined with Pph13-GAL4 resulted in the loss of the deep pseudopupil, as a readout for abnormal differentiation. In the end, we screened 6,189 lines, representing 3,971 genes, and have identified 64 genes, illuminating potential new regulatory molecules and cellular pathways for the differentiation and organization of Drosophila rhabdomeric photoreceptors.

Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum.

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3' UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR- mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.

The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila.

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.

A mosaic of independent innovations involving eyes shut are critical for the evolutionary transition from fused to open rhabdoms.

A fundamental question in evolutionary biology is how developmental processes are modified to produce morphological innovations while abiding by functional constraints. Here we address this question by investigating the cellular mechanism responsible for the transition between fused and open rhabdoms in ommatidia of apposition compound eyes; a critical step required for the development of visual systems based on neural superposition. Utilizing Drosophila and Tribolium as representatives of fused and open rhabdom morphology in holometabolous insects respectively, we identified three changes required for this innovation to occur. First, the expression pattern of the extracellular matrix protein Eyes Shut (EYS) was co-opted and expanded from mechanosensory neurons to photoreceptor cells in taxa with open rhabdoms. Second, EYS homologs obtained a novel extension of the amino terminus leading to the internalization of a cleaved signal sequence. This amino terminus extension does not interfere with cleavage or function in mechanosensory neurons, but it does permit specific targeting of the EYS protein to the apical photoreceptor membrane. Finally, a specific interaction evolved between EYS and a subset of Prominin homologs that is required for the development of open, but not fused, rhabdoms. Together, our findings portray a case study wherein the evolution of a set of molecular novelties has precipitated the origin of an adaptive photoreceptor cell arrangement.

Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates.

Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. We identify several hundred differentially expressed genes and dozens of differentially edited sites between flies from each slope, correlate these changes with genetic differences, and use CRISPR mutagenesis to validate that an intronic SNP in prominin regulates its editing levels. We also demonstrate that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences.

Two temporal functions of Glass: Ommatidium patterning and photoreceptor differentiation.

Much progress has been made in elucidating the molecular networks required for specifying retinal cells, including photoreceptors, but the downstream mechanisms that maintain identity and regulate differentiation remain poorly understood. Here, we report that the transcription factor Glass has a dual role in establishing a functional Drosophila eye. Utilizing conditional rescue approaches, we confirm that persistent defects in ommatidium patterning combined with cell death correlate with the overall disruption of eye morphology in glass mutants. In addition, we reveal that Glass exhibits a separable role in regulating photoreceptor differentiation. In particular, we demonstrate the apparent loss of glass mutant photoreceptors is not only due to cell death but also a failure of the surviving photoreceptors to complete differentiation. Moreover, the late reintroduction of Glass in these developmentally stalled photoreceptors is capable of restoring differentiation in the absence of correct ommatidium patterning. Mechanistically, transcription profiling at the time of differentiation reveals that Glass is necessary for the expression of many genes implicated in differentiation, i.e. rhabdomere morphogenesis, phototransduction, and synaptogenesis. Specifically, we show Glass directly regulates the expression of Pph13, which encodes a transcription factor necessary for opsin expression and rhabdomere morphogenesis. Finally, we demonstrate the ability of Glass to choreograph photoreceptor differentiation is conserved between Drosophila and Tribolium, two holometabolous insects. Altogether, our work identifies a fundamental regulatory mechanism to generate the full complement of cells required for a functional rhabdomeric visual system and provides a critical framework to investigate the basis of differentiation and maintenance of photoreceptor identity.

Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation.

BACKGROUND: Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure. RESULTS: We found that piperazine-N,N'-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging. CONCLUSIONS: We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research.

The actomyosin machinery is required for Drosophila retinal lumen formation.

Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.

Common transcriptional mechanisms for visual photoreceptor cell differentiation among Pancrustaceans.

A hallmark of visual rhabdomeric photoreceptors is the expression of a rhabdomeric opsin and uniquely associated phototransduction molecules, which are incorporated into a specialized expanded apical membrane, the rhabdomere. Given the extensive utilization of rhabdomeric photoreceptors in the eyes of protostomes, here we address whether a common transcriptional mechanism exists for the differentiation of rhabdomeric photoreceptors. In Drosophila, the transcription factors Pph13 and Orthodenticle (Otd) direct both aspects of differentiation: rhabdomeric opsin transcription and rhabdomere morphogenesis. We demonstrate that the orthologs of both proteins are expressed in the visual systems of the distantly related arthropod species Tribolium castaneum and Daphnia magna and that their functional roles are similar in these species. In particular, we establish that the Pph13 homologs have the ability to bind a subset of Rhodopsin core sequence I sites and that these sites are present in key phototransduction genes of both Tribolium and Daphnia. Furthermore, Pph13 and Otd orthologs are capable of executing deeply conserved functions of photoreceptor differentiation as evidenced by the ability to rescue their respective Drosophila mutant phenotypes. Pph13 homologs are equivalent in their ability to direct both rhabdomere morphogenesis and opsin expression within Drosophila, whereas Otd paralogs demonstrate differential abilities to regulate photoreceptor differentiation. Finally, loss-of-function analyses in Tribolium confirm the conserved requirement of Pph13 and Otd in regulating both rhabdomeric opsin transcription and rhabdomere morphogenesis. Taken together, our data identify components of a regulatory framework for rhabdomeric photoreceptor differentiation in Pancrustaceans, providing a foundation for defining ancestral regulatory modules of rhabdomeric photoreceptor differentiation.

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells.

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.

OTX2 and CRX rescue overlapping and photoreceptor-specific functions in the Drosophila eye.

BACKGROUND: Otd-related transcription factors are evolutionarily conserved to control anterior patterning and neurogenesis. In humans, two such factors, OTX2 and CRX, are expressed in all photoreceptors from early specification through adulthood and associate with several photoreceptor-specific retinopathies. It is not well understood how these factors function independently vs. redundantly, or how specific mutations lead to different disease outcomes. It is also unclear how OTX1 and OTX2 functionally overlap during other aspects of neurogenesis and ocular development. Drosophila encodes a single Otd factor that has multiple functions during eye development. Using the Drosophila eye as a model, we tested the ability of the human OTX1, OTX2, and CRX genes, as well as several disease-associated CRX alleles, to rescue the different functions of Otd. RESULTS: Our results indicate the following: OTX2 and CRX display overlapping, yet distinct subfunctions of Otd during photoreceptor differentiation; CRX disease alleles can be functionally distinguished based on their rescue properties; and all three factors are able to rescue rhabdomeric photoreceptor morphogenesis. CONCLUSIONS: Our findings have important implications for understanding how Otx proteins have subfunctionalized during evolution, and cement Drosophila as an effective tool to unravel the molecular bases of photoreceptor pathogenesis.