In Drosophila, the sine oculis (so) gene is important for the development of the entire visual system, including Bolwig's organ, compound eyes and ocelli. Together with twin of eyeless, eyeless, eyes absent and dachshund, sobelongs to a network of genes that by complex interactions initiate eye development. Although much is known about the genetic interactions of the genes belonging to this retinal determination network, only a few such regulatory interactions have been analysed down to the level of DNA-protein interactions. Previous work in our laboratory identified an eye/ocellus specific enhancer of the sine oculis gene that is directly regulated by eyeless and twin of eyeless. We further characterized this regulatory element and identified a minimal enhancer fragment of so that sets up an autoregulatory feedback loop crucial for proper ocelli development. By systematic analysis of the DNA-binding specificity of so we identified the most important nucleotides for this interaction. Using the emerging consensus sequence for SO-DNA binding we performed a genome-wide search and have thereby been able to identify eyeless as well as the signalling gene hedgehog as putative targets of so. Our results strengthen the general assumption that feedback loops among the genes of the retinal determination network are crucial for proper development of eyes and ocelli.

The homeobox gene orthodenticle (otd) controls the process of regional specification that takes place in the Drosophila eye-antennal disc during ocelli development. Mutations that reduce or abolish otd expression in the ocelli primordium give rise to ocelliless flies. We have identified the cis-regulatory sequence (ocelliless enhancer) that controls otd expression during ocelli development and studied its regulation at the molecular level. The ocelliless enhancer is initially activated by the combined action of Wingless (Wg) and Hedgehog (Hh) signaling pathways. Later, a positive autoregulatory feedback loop sets in to maintain otd expression. Moreover, we have analyzed the role of otd during ocelli primordium development and determined its involvement in the expression of the retinal determination gene eyes absent (eya). otd indirectly regulates eya in ocellar precursor cells through the inhibition of wg, an eya repressor, and the maintenance of hh expression in the ocelli primordium. Hh signaling is necessary for eya activation in ocellar precursor cells and this activation is mediated by the full-length activator form of the transcription factor Cubitus interruptus.

The homeobox gene sine oculis (so) is required for the development of the entire visual system in Drosophila, which includes the compound eyes, the ocelli, the optic lobe of the brain and the Bolwig's organ. During ocelli development, so expression labels, together with eyes absent (eya), the emergence of the ocellar precursor cells in the third instar eye-antennal disc. Footprinting and misexpression studies have led to the proposal that the Pax6 homologue twin of eyeless (toy) directly regulates the initiation of so expression in ocellar precursor cells. However, so expression in a toy loss-of-function mutant background has not been yet analyzed due to the lack of eye-antennal disc development in strong toy mutant alleles. Using an embryonic eye primordium-specific enhancer of toy, we have rescued the developmental defect of a strong toy mutant allele and analyzed so expression in the ocelli primordium of toy loss-of-function eye-antennal discs during third instar larva. The results show that so expression is only marginally affected in the absence of Toy transcriptional activity and that the toy positive effect on so expression is largely eya-mediated. These results suggest that eya is the main factor controlling both initiation and maintenance of so expression in ocellar precursor cells. In addition, we present the characterization of a new minimal eye/ocellus-specific enhancer of the so gene.

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