Identification of a Limb‐Specific Sonic Hedgehog Regulatory Module

Sonic hedgehog (Shh) is a potent developmental morphogen, pivotal in the growth and patterning of many organ systems. Regulation of its limb‐specific expression within the zone of polarizing activity (ZPA) in the posterior limb margin is crucial for normal limb development. Ectopic limb expression of Shh causes polydactyly. Absence of limb‐related Shh expression causes adactyly. Both defects can be linked to mutations in an intron of the Lmbr1 gene, 1Mb upstream of Shh . We have identified evolutionarily conserved regions (ECRs) across divergent species within this limb‐specific Shh regulatory region (LSSRR). These ECRs likely contain the regulatory modules that coordinate limb‐specific Shh expression. A 1.8kb DNA fragment of the LSSRR was isolated and ECR peaks were identified as Peak A and B; Peak B was subdivided into B1, B2, and B3. Enhancer‐reporter constructs were generated by inserting the LSSRR or individual ECR fragments upstream of a minimal HSV‐tk promoter linked to green fluorescent protein (GFP). The constructs were co‐electroporated with a red fluorescent protein (RFP) reporter to verify transfection efficiency. Enhancer activation was marked by localized GFP expression. Embryos were monitored for fluorescence and harvested between 36–48 hrs. Broad GFP expression in the posterior half of the limb, coincident with cells that had expressed Shh, was detected with LSSRR, peak B or peak B3 (321 bp). In addition, when B3 was mutated at a potential binding site for NFκB, a transcription factor linked to Shh regulation, GFP expression was markedly reduced. Our data suggests that ECR Peak B3 contains a limb‐specific regulatory module for Shh expression. Further experiments are needed to characterize this module and verify whether NFκB is, in part, responsible for its enhancer activity.