Intronic enhancers control expression of zebrafish sonic hedgehog in floorplate and notochord

The signalling molecule Sonic hedgehog (Shh) controls a wide range of diffe rentiation processes during vertebrate development, Numerous studies have s uggested that the absolute levels as well as correct spatial and temporal e xpression of shh are critical for its function. To investigate the regulati on of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish, We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences wit h a reporter construct and analysis of mosaic expression in accumulated exp ression maps. Enhancers were identified in intron 1 and 2 that mediate floo r plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mec hanisms controlling shh expression in the midline are conserved between zeb rafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enha ncers have a complex organisation. Two activator regions, ar-A and ar-C, we re identified in intron 1 and 2, respectively, which mediate mostly notocho rd and floor plate expression. In contrast, another activating region, ar-B , in intron 1 drives expression in the floor plate. Deletion fine mapping o f ar-C delineated three regions of 40 bp to be essential for activity. Thes e regions do not contain binding sites far HNF3 beta, the winged helix tran scription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcript ion factor-binding site was found in a functionally important region that f orms specific complexes with protein extracts from wild-type but not from n otochord-deficient mutant embryos.