Fig. 1. Conservation of Shh regulatory sequences across phyla. (A) A schematic of the Shh locus showing the location of coding exons (black boxes), noncoding sequences (solid line) and intronic enhancers (gray oval, Sbe1; yellow oval, Sfpe2). The sequence of the 746 bp region of intron 2 overlapping Sfpe2 activity in the mouse was compared with human, chicken and zebrafish, identifying three regions of high sequence homology corresponding to homology region-a (HR-a, blue), HR-b (green) and HR-c (red). (B) Reporter constructs (RC) designed to assay Sfpe2 activity. DNA segments from the 746 bp region were cloned downstream of a reporter cassette containing a minimal Shh promoter (Ius), lacZ gene (light-blue box) and Sbe1 sequences (gray oval). To the right of the constructs are the results of the transgenic expression analysis indicating the total number of transgenic embryos generated for each construct (#Tg) and the number of embryos that stained in the ventral midline of the spinal cord (SC) and midbrain (MB). The consistency of staining intensity and minimal time to initiate staining were used to subjectively classify the spinal cord expression as strong (+++), moderate (++), weak and patchy (+/–), or absent (–). The lacZ expression generated in the midbrain is regulated by Sbe1 and serves as a positive control for transgene expression. Rc1 and Rc2 were reported previously (Epstein et al., 1999). (C) X-gal staining of transgenic embryos carrying reporter constructs at 9.5 dpc. The cranial staining depicted by the embryo carrying Rc1 is also representative of the staining pattern seen in embryos expressing Rc3-6.

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