Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates.In the developingDrosophilaeye, 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 discovered 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 homologues Scratch and Scrape, as well as directly activating neuronal 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 gl60j mutant scRNA-Seq deposited here was performed in Drosophila melanogaster on white prepupal stage eye discs. The corresponding control scRNA-Seq experiment with wild type white prepupal eye discs was published at https://doi.org/10.1038/s41467-023-43037-0 and the raw data deposited at GSE235110. The DamID experiment was performed on wandering 3rd instar larval eye discs for two transcription factors, Gl and Pnt (PntP1 isoform), as well as Dam-only controls. The GAL4 drivers used were atonal-GAL4, which is expressed in the morphogenetic furrow, and elav-GAL4, which is expressed in the committed photoreceptors. All Dam-ID experiments were performed in triplicate biological replicates. RNA-Seq was carried out on wandering third instar larval wing discs that contained clones expressing GFP; GFP and Gl; GFP and RasV12; GFP, Gl and RasV12; GFP, Gl and RasV12 in pntdelta88 mutant clones; and GFP and Gl in cicQ219X mutant clones. The goal was to discover genes that are synergistically activated by Gl and EGFR signaling. This bulk RNA-Seq experiment was performed in triplicate biological replicates. We perfomed RNA-Seq analysis on temperature sensitive mutant egfrtsla/egfrf2 wandering 3rd instar larval eye discs. Control samples were kept at the permissive temperature of 18 degrees C and mutant samples were shifted to the restrictive temperature of 29 degrees C for 24 h. This bulk RNA-Seq experiment was performed in triplicate biological replicates.