Regulation of Sonic hedgehog (Shh) expression in the zebrafish retina

Sonic hedgehog (Shh) a vertebrate homolog of the Drosophila melanogaster gene hedgehog, is a secreted protein that controls numerous differentiation processes during vertebrate development. In the vertebrate retina, Shh controls neurogenesis and its expression spreads in a wave like manner. The shh locus has been well characterised in vertebrates and the regulatory regions driving the expression in the notochord and the midline of the CNS were identified. In zebrafish, shh transgenes were established that drive GFP reporter expression very similar to that of the endogenous shh gene in the central nervous system including the retina. The cis-regulation behind shh expression in the retina was not known. This prompted me to map the region responsible for retina expression. I found it to be distinct from the previously described enhancers controlling expression in the midline of the neural tube and in the notochord. This novel regulatory region mediates expression of a GFP reporter cassette in the ganglion cell layer (GCL) and inner nuclear layer (INL) of the retina. The expression is initiated in a ventronasal patch and later spreads across the GCL and INL in a wave like pattern. A deletion approach identified a 300 bp region to be sufficient and necessary for driving expression in the retina. By a second series of mutations across this region using a linker scanning approach, a minimal 40 bp core important for expression was identified. While one clusters of point mutation impaired expression in both GCL and INL, another region was mapped that affected expression exclusively in the GCL. Thus, expression in the two layers can be separated. Pea3 and Erm factors were predicted to bind to the retinal enhancer. In vitro protein binding and morpholino studies prove that they are key regulatory factors responsible for driving expression in the retina. Pea3 and Erm are factors known to act downstream of Fgf signals. Earlier studies have demonstrated that Fgf signalling from t