Cross-species transcriptomics and motif discovery reveal a conserved Glass targetome in Drosophila eye development

Changes in developmental gene regulatory networks are considered as important drivers of diversity, yet fundamental core networks are often conserved in evolution. Our aim is to study conserved and divergent interactions in the gene regulatory network (GRN) underlying photoreceptor specification, downstream of the retinal determination network operated by toy, ey, so, eya, and dac. To this end, we performed a comparative genome-wide expression study in eye-antennal and wing imaginal discs across three Drosophila species, namely D.melanogaster, D.yakuba and D.virilis using Tag-Seq profiling. By integrating expression ratios of the three species with order statistiscs we identified a highly conserved core of genes enriched in compound eye photoreceptor development. Next, we performed cis-regulatory sequence analysis on this gene set, across 12 Drosophila species. For this, we used the method i-cisTarget and found the Glass position weight matrix (PWM) as the highest ranked motif within a library of more than 6000 PWMs. The Glass motif predicted 95 conserved direct Glass targets including lz – its only previously known target. To validate the Glass target predictions, we performed differential expression analysis between wild-type and glass mutant discs using RNA-Seq experiment. This showed that 72 (76%) of the predicted target genes are significantly differentially expressed. Finally, we investigated the predicted target enhancers and observed that in most of the cases there is only one high Glass scoring regulatory region conserved across species. Interestingly, each target presents several regions enriched in a species-specific manner. To validate the predicted Glass binding regions in eye development we assessed their enhancer activity taking advantage of available D.melanogaster enhancer-GAL4 lines and the GAL4-UAS system in Drosophila. Seven out nine tested conserved enhancers presented expression and overlapped with Glass eye expression in D.melanogaster, yielding a 78 % of success rate. To study the conservation of the regulatory activity in other Drosophila species we also cloned several D.virilis orthologous enhancers. Currently, three enhancers have been tested, one in chp and two in scrt genes confirming functional conservation across evolution. Finally, we have predicted a D.virilis specific enhancer in the Glass target gene scrt, for which the D.melanogaster orthologous region does not drive eye expression in eye development.status: publishe