FGF‐INDUCED ACTIVATION OF THE LIMB SPECIFIC SHH REGULATORY REGION (LSSRR)

Abstract

Regeneration of amputated embryonic chick wings can be induced by Fgf, and upregulation of Shh is an essential step in this process. During development, a 1.8 kb conserved non‐coding region up‐stream of Shh functions as a limb‐specific Shh regulatory region (LSSRR). We hypothesize that LSSRR is involved in Fgf‐induced limb regeneration. To test our hypothesis, we electroporated (EP) an LSSRR‐green fluorescent protein (GFP)‐reporter construct into stage 23 chick limb bud mesoderm and then implanted an Fgf‐soaked bead to induce Shh expression. Green fluorescence was detected around the Fgf bead after 24 hr, supporting a role for LSSRR in the up‐regulation of Shh by Fgf. To determine the sequences within the LSSRR that were required for Shh up‐regulation, we isolated three separate candidate sequences within the LSSRR that contained Hox or RAR/NFκB binding site. The candidate sequences were ligated into a ptk‐GFP reporter plasmid and then EP into the presumptive wing mesoderm of stage 14 chick embryos. All three candidate sequences showed fluorescence at 48 hr, indicating enhancer activity with these minimal fragments. We conclude that during chick wing regeneration, Fgf utilizes the LSSRR in upregulating Shh and that multiple enhancer elements within the LSSRR may contribute to Shh regulation.Grant Funding Source : Departmental Funding