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The  Drosophila  eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. Here, we report transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. Our data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as well as early cone cells. We identify dozens of cell type-specific genes whose function in different aspects of eye development have not been reported. These single cell data will greatly aid research groups studying different aspects of early eye development and will facilitate a deeper understanding of the larval eye as a model system.

The Drosophila eye is a powerful model system to study the dynamics of cell differentiation, cell state transitions, cell maturation, and pattern formation. However, a high-resolution single cell genomics resource that accurately profiles all major cell types of the larval eye disc and their spatiotemporal relationships is lacking. Here, we report transcriptomic and chromatin accessibility data for all known cell types in the developing eye. Photoreceptors appear as strands of cells that represent their dynamic developmental timelines. As photoreceptor subtypes mature, they appear to assume a common transcriptomic profile that is dominated by genes involved in axon function. We identify cell type maturation genes, enhancers, and potential regulators, as well as genes with distinct R3 or R4 photoreceptor specific expression. Finally, we observe that the chromatin accessibility between cones and photoreceptors is distinct. These single cell genomics atlases will greatly enhance the power of the Drosophila eye as a model system. The Drosophila eye is a powerful model system to study the dynamics of cell differentiation, cell state transitions, cell maturation, and pattern formation. Here, the authors report transcriptomic and chromatin accessibility data for all known cell types in the developing larval eye.

SPATA7 , an early onset LCA3 retinal disease gene, encodes a putative scaffold protein that is essential for the proper assembly of the connecting cilium (CC) complex in photoreceptors. Previous studies have shown that SPATA7 interacts with other photoreceptor-specific ciliary proteins, such as RPGR and RPGRIP1, and maintains the integrity of CC integrity. However, although it is known that Spata7 is required for early formation of the CC, it is unclear if Spata7 is also required for the maintenance of the CC. To investigate Spata7 function in the retina at the adult stage, loss of function was induced in the adult retina upon tamoxifen induction of an inducible Spata7 knockout allele ( Spata7 flox/− ; UbcCreERT2/ + ). The phenotype of mutant retina was characterized by a combination of histology, immunobiochemistry, and electroretinography (ERG). Our results demonstrated that Spata7 is also essential for maintaining the integrity of the mature retinal CC. Loss of Spata7 in adults caused phenotypes similar to those seen in germline mutant mice, including photoreceptor cell degeneration and defective ERG responses. Close examination of the CC revealed significantly shortened NPHP1 length as a result of Spata7 deletion. Furthermore, mislocalization of rhodopsin, leading to ER stress-mediated apoptosis, was observed in the retinal layers. Our results indicate that Spata7 is required not only for the establishment but also for the maintenance of the CC of photoreceptors.

The Ets domain transcription factors direct diverse biological processes throughout all metazoans and are implicated in development as well as in tumor initiation, progression and metastasis. The Drosophila Ets transcription factor Pointed (Pnt) is the downstream effector of the Epidermal growth factor receptor ( Egfr ) pathway and is required for cell cycle progression, specification, and differentiation of most cell types in the larval eye disc. Despite its critical role in development, very few targets of Pnt have been reported previously. Here, we employed an integrated approach by combining genome-wide single cell and bulk data to identify putative cell type-specific Pnt targets. First, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine the genome-wide occupancy of Pnt in late larval eye discs. We identified enriched regions that mapped to an average of 6,941 genes, the vast majority of which are novel putative Pnt targets. Next, we integrated ChIP-seq data with two other larval eye single cell genomics datasets (scRNA-seq and snATAC-seq) to reveal 157 putative cell type-specific Pnt targets that may help mediate unique cell type responses upon Egfr-induced differentiation. Finally, our integrated data also predicts cell type-specific functional enhancers that were not reported previously. Together, our study provides a greatly expanded list of putative cell type-specific Pnt targets in the eye and is a resource for future studies that will allow mechanistic insights into complex developmental processes regulated by Egfr signaling.

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. The authors show that synergistic activation by two transcription factors - Pointed, which is activated by cell–cell signaling, and Glass, which is eye-specific - drives a program of neuronal gene expression in developing Drosophila photoreceptors.

Tubulogenesis is essential for the formation and function of internal organs. One such organ is the trachea, which allows gas exchange between the external environment and the lungs. However, the cellular and molecular mechanisms underlying tracheal tube development remain poorly understood. Here, we show that the potassium channel KCNJ13 is a critical modulator of tracheal tubulogenesis. We identify Kcnj13 in an ethylnitrosourea forward genetic screen for regulators of mouse respiratory organ development. Kcnj13 mutants exhibit a shorter trachea as well as defective smooth muscle (SM) cell alignment and polarity. KCNJ13 is essential to maintain ion homeostasis in tracheal SM cells, which is required for actin polymerization. This process appears to be mediated, at least in part, through activation of the actin regulator AKT, as pharmacological increase of AKT phosphorylation ameliorates the Kcnj13- mutant trachea phenotypes. These results provide insight into the role of ion homeostasis in cytoskeletal organization during tubulogenesis. Tubulogenesis is required for the formation of many internal structures including the trachea. Here, the authors show that the potassium channel KCNJ13 regulates tracheal tube formation, with shorter tracheas forming in mutant mice due in part to changes in actin organization in tracheal smooth muscle cells.

Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the \"retinal determination gene network,\" composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.

The adult Drosophila eye is a powerful model system for phototransduction and neurodegeneration research. However, single cell resolution transcriptomic data are lacking for this tissue. We present single cell RNA-seq data on 1-day male and female, 3-day and 7-day old male adult eyes, covering early to mature adult eyes. All major cell types, including photoreceptors, cone and pigment cells in the adult eye were captured and identified. Our data sets identified novel cell type specific marker genes, some of which were validated in vivo. R7 and R8 photoreceptors form clusters that reflect their specific Rhodopsin expression and the specific Rhodopsin expression by each R7 and R8 cluster is the major determinant to their clustering. The transcriptomic data presented in this report will facilitate a deeper mechanistic understanding of the adult fly eye as a model system. Single-cell RNA sequencing of early to mature adult Drosophila eyes reveals new cell type-specific marker genes and a full set of transcriptomic data for this tissue.

Skin lightening among Eurasians is thought to have been a convergence occurring independently in Europe and East Asia as an adaptation to high latitude environments. Among Europeans, several genes responsible for such lightening have been found, but the information available for East Asians is much more limited. Here, a genome-wide comparison between dark-skinned Africans and Austro-Asiatic speaking aborigines and light-skinned northern Han Chinese identified the pigmentation gene OCA2, showing unusually deep allelic divergence between these groups. An amino acid substitution (His615Arg) of OCA2 prevalent in most East Asian populations—but absent in Africans and Europeans—was significantly associated with skin lightening among northern Han Chinese. Further transgenic and targeted gene modification analyses of zebrafish and mouse both exhibited the phenotypic effect of the OCA2 variant manifesting decreased melanin production. These results indicate that OCA2 plays an important role in the convergent skin lightening of East Asians during recent human evolution.

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions.