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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., (© 2024. The Author(s).)

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., (Copyright © 2016 Iyer et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

The eye-antennal imaginal disc of Drosophila melanogaster has often been described as an epithelial monolayer with complex signaling events playing out in two dimensions. However, the imaginal disc actually comprises two opposing epithelia (the peripodial epithelium, or PE, and the disc proper, or DP) separated by a lumen to form a sac-like structure. Recent studies expose complex molecular interactions between the PE and the DP, and reveal dynamic communication between the two tissues. Further findings suggest the PE makes important contributions to DP development by acting as a source of signaling molecules as well as cells. Here we summarize those findings and highlight implications for further research., (2009 Wiley-Liss, Inc.)

Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens.

Drosophila dachshund is a critical regulator of eye, brain, and limb formation. Vertebrate homologs, Dach1 and Dach2, are expressed in the developing retina, brain, and limbs, suggesting functional conservation of the dachshund/Dach gene family. Dach1 mutants die postnatally, but exhibit grossly normal development. Here we report the generation of Dach2 mutant mice. Although deletion of Dach2 exon 1 results in abrogation of RNA expression, Dach2 mutants are viable and fertile. Histochemical analysis reveals grossly normal Dach2 mutant eye development. In addition, a battery of neurological assays failed to yield significant differences in behavior between Dach2 mutants and controls. We discuss these findings in the light of published observations of DACH2 mutations in the human population. Finally, to test the functional conservation hypothesis, we generated Dach2; Dach1 double mutant mice. Dach double mutants die after birth, similar to Dach1 homozygotes. However, unlike Drosophila dachshund mutants that lack eyes and exhibit leg truncations, the eyes and limbs of Dach double mutants are present, suggesting differences between Dach and dachshund gene function during embryonic eye and limb formation.

With its unique structure and dynamic development, the Drosophila eye has been a powerful genetic model system for studying molecular mechanisms of cell fate specification and differentiation. Hundreds of genes that function in a complex genetic network controlling this process have been identified during the past two decades. To further advance our understanding of the molecular mechanisms of eye development, it is increasingly important to place the current genetic pathway into a whole-genome perspective. Here, we review emerging technologies and strategies that will help to achieve this goal, including generation of a complete mutant set in Drosophila, genome-wide transcription factor target identification, and systematic studies of gene function aided by computational biology.

The possibility that mechanisms of retinal determination may be similar between vertebrates and Drosophila has been supported by the observations that Pax6/eyeless genes are necessary and sufficient for retinal development. These studies suggest that the function of other gene families, operating during early eye development, might also be conserved. One candidate is the retinal homeobox (Rx) family of transcription factors. Vertebrate Rx is expressed in the prospective eye and forebrain and is required for eye morphogenesis, retinal precursor appearance, and normal forebrain development, indicating that it is an essential regulator of early eye and brain formation. Here, we test the hypothesis that Drosophila Rx (drx) is required for adult and larval eye development. We have isolated a drx null allele and demonstrate that the mutant compound eye and larval visual system is not detectably abnormal. However, we find that drx is required for development of a central brain structure, the ellipsoid body, suggesting that Rx function in the brain may be conserved. Finally, we characterize a novel anterior head phenotype and demonstrate that drx is required for clypeus development. Thus, our data suggest that drx may be required for the regulation of genes involved in brain morphogenesis and clypeus precursor development. We propose that differences in insect and vertebrate eye development may be explained by changes in gene regulation and/or the tissue of origin for eye precursor cells.

The Drosophila eye is an outstanding model with which to decipher mechanisms of neural differentiation. Paramount to normal eye development is the organized selection and differentiation of a patterned array of R8 photoreceptors - the founding photoreceptor of each ommatidium that coordinates the incorporation of all other photoreceptors. R8 development is a complex process that requires the integration of transcription factors and signaling pathways, many of which are highly conserved and perform similar functions in other species. This article discusses the developmental control of the four key elements of R8 development: selection, spacing, differentiation and orchestration of later events. New questions that have surfaced because of recent advances in the field are addressed, and the unique characteristics of R8 development are highlighted through comparisons with neural specification in other Drosophila tissues and with ganglion cell development in the mammalian retina.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

Drosophila dachshund is necessary and sufficient for compound eye development and is required for normal leg and brain development. A mouse homologue of dachshund, Dach1, is expressed in the developing retina and limbs, suggesting functional conservation of this gene. We have generated a loss-of-function mutation in Dach1 that results in the abrogation of the wild-type RNA and protein expression pattern in embryos. Homozygous mutants survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress. The heart, lungs, kidneys, liver, and skeleton were examined to identify factors involved in postnatal lethality, but these organs appeared to be normal. In addition, blood chemistry tests failed to reveal differences that might explain the lethal phenotype. Gross examination and histological analyses of newborn eyes, limbs, and brains revealed no detectable abnormalities. Since Dach1 mutants die shortly after birth, it remains possible that Dach1 is required for postnatal development of these structures. Alternatively, an additional Dach homologue may functionally compensate for Dach1 loss of function.

Neural specification and differentiation in the Drosophila eye sweep across the unpatterned epithelial monolayer of the eye imaginal disc following a developmental wave termed the morphogenetic furrow. The furrow begins at the posterior margin of the eye imaginal disc and moves anteriorly as a linear front. Progression of the furrow requires the function of hedgehog, which encodes a secreted signaling protein. We characterize mutations in dachshund, a gene that encodes a novel nuclear protein required for normal cell-fate determination of imaginal disc cells. In the absence of dachshund function, cells at the posterior margin of the eye disc fail to follow a retinal differentiation pathway and appear to adopt a cuticle fate instead. These cells are therefore unable to respond to pattern propagation signals such as hedgehog and furrow initiation does not occur. In contrast, cells in more anterior portions of the eye disc are able to differentiate as retinal cells in the absence of dachshund activity and respond normally to patterning signals. These results suggest that posterior margin cells are distinct from other cells of the eye imaginal disc by early stages of development. dachshund is also necessary for proper differentiation of a subset of segments in the developing leg. Null mutations in dachshund result in flies with no eyes and shortened legs.