Functional Dissection Of EYES ABSENT And SINE OCULIS In Drosophila Eye Development

Eye formation depends on the retinal determination network genes (RDGN), which encode highly conserved transcription factors and cofactors that are necessary for eye formation. Eyes absent (Eya) and Sine oculis (So) form a regulatory complex that directs developmental processes, including cell fate, differentiation, proliferation, and survival in organisms ranging from flies to humans. Eya is a protein phosphatase and transcriptional coactivator, whereas its binding partner So is a DNA-binding transcription factor. In Drosophila, eya and so are necessary for survival, as well as being both necessary and sufficient for eye formation. Overexpression of them can cause ectopic eye induction. Despite their vital roles in metazoan development, much remains to be learned about the mechanisms of Eya and So regulation and function in vivo. In the current studies, we set out to expand our understanding of Eya and So with two specific aims:structure-function analysis of Eya in the eye by using genomic rescue system, and identification of novel transcriptional targets of So by using ChIP-seq. Research results as follows:First, tyrosine phosphatase of Eya is not required for development or survival in Drosophila. Drosophila eya encodes three protein isoforms, all three isoforms share two main functional domains:the highly conserved271-amino acid C-terminal domain, known as Eya Domain1(ED1), and the moderately conserved Eya Domain2(ED2), embedded in a216-amino acid proline-serine-threonine rich (PST) region. EDI contains tyrosine phosphatase activity, in vitro phosphatase assays show that Eya proteins derived from plant, mouse, and fly exhibit tyrosine phosphatase activity, although that of Drosophila Eya is very low and is difficult to detect. Multiple lines of evidence suggest that Eya tyrosine phosphatase regulates development in mammals. In Drosophila, cDNA-based eya mutant transgenes that disrupt the predicted tyrosine phosphatase active site have drastically decreased ability to induce ectopic eye formation and to rescue eye development in the eya2mutant. In addition, a study based on overexpression of wild type and phosphatase-inactive eya transgenes in the developing eye suggests that Eya tyrosine phosphatase regulates photoreceptor axon targeting. Although these findings suggest that Drosophila Eya tyrosine phosphatase activity may play a role during normal development, this hypothesis has not been tested using a system that accurately reproduces endogenous levels, timing, and patterns of 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 constructs that alter key active-site residues. We used a two-step recombineering method to create the D493-N (GAT->AAT) and E728-Q (GAG->CAG) point mutations in the eya+GR construct. Two of the point-mutant constructs, eyaDD493NGR and eyaE728QGR, encode a protein with a single amino-acid substitution:D493N and E728Q, respectively. In the third construct, eyaNQGR, we engineered both D493N and E728Q mutations in eya+GR. Hereafter eyaD493NGR, eyaE728QGR and eyaNQGR are collectively described as eya*GR. We then employed cpC31to integrate constructs into attP2on the third chromosome and test if they can resuce eya eye-specific mutant eya2and null mutant eyacliHD/Df(2L)BSC356in vivo. In striking contrast to previous studies, all eya*GR constructs fully restore eye formation as well as viability in eya mutants; rescued flies show normal fertility, viability, external and internal eye morphology, eye disc development, response to light and photoreceptor axon targeting.Previous studies have reported structure-function analysis of Eya by cDNA-based transgene (ectopic eye induction and heat-shock induction), this approach has several drawbacks, including inaccurate timing, patterns, and levels of transgene, while our genomic rescue constructs are likely to contain the full complement of regulatory sequences needed, allowing the transgene to be expressed in the same spatiotemporal pattern as the endogenous gene of interest. Our study suggests the need for re-evaluation of the mechanism of Eya action, underscores the importance of studying genes in their native context and will fundamentally change the way the field will view this highly conserved protein.Second, transactivation domain of eya is required for development and survival in Drosophila. Eya PST domain contains the ED2domain and two MAPK target sites. The ED2domain has threonine phosphatase, which is in the middle of the PST region. The threonine phosphatase activity of Eya regulates the innate immune response in both mammals and flies, and it is currently not known to have a function in development. Recent in vivo studies have reported that the MAPK-mediated phosphorylation does not regulate Eya during normal Drosophila development and survival. cDNA-based studies have also suggested that an internal PST domain in Eya is required for transcriptional activation in cell culture reporter assays and for efficient induction of ectopic eyes in vivo. Although PST domain plays a vital role in transactivition function of Eya and deletion will cause low efficiency of ectopic eye induction, this hypothesis has not been tested using a system that accurately reproduces endogenous levels, timing, and patterns of eya expression.To investigate whether the PST tansactivation domain of Eya plays a role in Drosophila survival or normal eye formation, we used a one-step recombineering method to create the deletion mutation in the eya+GR construct, eya△PSTGR. We then employed φC31to integrate eya△PSTGR construct into attP2on the third chromosome and test if it can rescue Drosophila development and survival in eya mutant background. Our results form genomic rescue system and FRT-FLP mosaic clonal analysis suggest that eya△PSTGR fail to rescue eya eye-specific mutant and null mutant. RT-PCR and Western Blot reveal that eya△PST have expression on the transcriptional and protein levels. Immnohistochemistry detection earlier, the protein expression level is closer to the wild type. All of the above suggest rescue failure is not caused by no eya△PST protein expression, but due to the deletion of PST domain.Third, So regulates multiple genes in Drosophila eye development. Among the best-characterized binding partners of So is the transcriptional coactivator and protein phosphatase Eya. So is the founding member of the SIX (Sine oculis homeobox) family of homeodomain transcription factors, which occur in all metazoans and are characterized by two conserved domains:the119-amino acid SIX Domain (SD), which mediates protein-protein interactions, and the60-amino acid DNA-binding homeodomain (HD). In Drosophila, So is required for survival, spermatogenesis and the development of the entire visual system, including the eyes, the optic lobes of the brain, and Bolwig’s organ. Despite the vital role of So in eye development, only a few So targets have been identified to date. Most of these targets encode transcriptional regulators necessary for various stages of eye development.In order to improve our understanding of how So regulates eye development, we have performed chromatin immunoprecipitation with an anti-So antibody followed by genome-wide next-generation sequencing (ChIP-seq) on third instar eye-antennal discs. We found7566So-enriched regions (peaks) throughout the genome, corresponding to approximately5400genes. The So-enriched gene set includes previously characterized direct targets of So. We selectd a few genes and tested them by qPCR, results show So enrichment. Genes enriched for highly significant So peaks are over-represented in Gene Ontology categories that pertain to eye development. We have obtained mutant alleles of a subset of So-bound, novel genes, and have assayed their function in the eye. Finally, we identified eight novel genes that are necessary for eye development and may act downstream of So.The eight mutants show a range of phenotypes, from a rough but full-sized eye to almost complete loss of eye, reflecting the requirement of So at multiple stages of eye development, from cell survival and MF initiation to differentiation. The genes are predicted to encode two RNA processing proteins (omd and Syp); two putative transcription factors (CG8108and l(3)j2D3); a RabGGT subunit, which regulates intracellular vesicle trafficking (CG12007); a transmembrane transporter (blot); and two proteins of unknown function, with an Armadillo-like fold (CG2747) and WD40repeat (CG13192). Our results greatly expand the set of putative So targets in the developing eye, and set the stage for many future studies of So function in development.