Interaction of fibroblast growth factor and Sonic Hedgehog signaling in limb development

Fibroblast growth factor (Fgf) signaling from the Apical Ectodermal Ridge (AER) is essential for inducing and maintaining the expression of Sonic Hedgehog (Shh) in the Zone of Polarizing Activity (ZPA) of the posterior limb bud, which in turn patterns the anterior-posterior (A-P) axis of the limb and stimulates AER-Fgf expression. Etv genes encode ETS-domain containing transcription factors downstream of Fgf pathway. To address whether they mediate AER-Fgf signals for proximal-distal (P-D) outgrowth and Shh expression, I knocked out both Etv4 and Etv5, the only two Etv genes expressed in limb bud, specifically in limb bud mesenchyme. This inactivation led to ectopic Shh expression in the anterior limb bud, and a preaxial polydactyly (PPD) skeletal phenotype, but not to defects in P-D outgrowth. The phenotypes suggest that these transcription factors inhibit Shh expression in the anterior limb bud. This finding suggests that FGF signaling facilitates the ability of SHH to polarize the limb bud by both promoting and restricting Shh expression.;In addition to Etv genes, I investigated the role of Twist1, a known Shh repressor, in mouse limb development. Previous studies based on conventional knockout phenotypes suggested that Twist1 is essential to maintain AER-Fgfs/Shh and Fgf8/Fgf10 loops during mouse limb development. By analyzing the phenotypes of conditional knockout mutants, I found that Twist1 is not required to maintain those loops. Moreover, my data support novel roles of Twist1 in the survival of AER precursors and girdle patterning, and suggested that Twist1 functions as a major Shh repressor compared to others.;The similar expression domain and knockout phenotype of Etv genes and Twist1 suggest that they may interact in regulating Shh expression. By generating Etv and Twist1 compound mutants, I demonstrated that they act synergistically to regulate Shh, and function through the same pathway. Additionally, I provided genetic evidence that like Twist1, Etv genes antagonize Hand2, a key activator of Shh. Furthermore, I found that ETV5 protein interacts with the TWIST1 and HAND2 proteins in vivo. My findings suggest a molecular mechanism by which Etv genes function as Twist1 modulator to repress Shh.