During limb development, fibroblast growth factors (Fgfs) govern proximal–distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal–distal and anterior–posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.

Shh is critical to limb patterning during development and regeneration. The apical ectodermal ridge secretes Fgfs that induce limb outgrowth and maintain Shh expression in the zone of polarizing activity through a reciprocal Fgf‐Shh loop. Gene arrays performed following FGF2 application to the posterior region of the chick limb bud demonstrate elevated levels of WNT5A, FZD4, DKK1, and SHH. We hypothesized that the Wnt pathway might participate in the Fgf‐induced up‐regulation of Shh.Thus, we analyzed the expression time course of WNT5A, FZD4, and DKK1 during FGF‐induced SHH up‐regulation. FGF2 soaked‐beads were implanted into the posterior aspect of stage 23 chick wing buds; the chicks were then harvested after 3, 6, 12, 18, and 24 hr of incubation and performed in situ hybridization for SHH and the Wnt‐associated genes.SHH was up‐regulated by FGF2 within 3 hr of application. DKK1 was the first of the Wnt‐associated genes to be up‐regulated by FGF2, 3 hr after application followed by WNT5A at 12 hr, and then FZD4 at 18 hr.This sequence of expression was unexpected. It was anticipated that up‐regulation of WNT5A by FGF2 would regulate SHH. However, up‐regulation of SHH precedes WNT5A. It is possible that the Wnt pathway is activated by FGF2 in a coincident, but separate pathway from SHH up‐regulation. It is also feasible that DKK1 could up‐regulate SHH and further studies are needed to investigate this possibility.