Figure 1.

Strategy to find direct targets of Ey. (A) The Drosophila retinal determination network. Retinal cell fate is established by an interregulatory network of conserved nuclear factors. toy induces ey, which induces so and eya, which induce dac. This pathway receives input from secreted signals from the hh and dpp pathways, which promote retinal determination, and the wg pathway that inhibits it. Several other genes are necessary and/or sufficient for eye development, including Optix and eyegone (eyg). These factors establish competency for the next stage of development, photoreceptor differentiation, whose first step, R8 recruitment, requires genes such as ato and sens. (B) Genes that upregulated by ey were identified by conducting microarray analysis of wild-type versus dpp-GAL4/UAS-ey discs. Quantitative real-time RT-PCR and hand curation through comparison to online databases were used to evaluate the results. We searched for possible Ey binding sites in this list of 188 genes, identifying 20 putative direct targets. Expression pattern and response to ey was examined by in situ hybridization and RT-PCR. In vitro Ey binding was tested by EMSAs. Reporter analysis of fragments containing identified Ey binding sites was used to test their function in vivo.