LHX2: a critical link in the FGF to SHH pathway? (541.2)

Abstract

Fibroblast growth factors (FGFs) and Sonic hedgehog (SHH) are morphogens known to be integrally involved in limb outgrowth/development. Published reports assert that for SHH to be expressed, FGFs have to be secreted from the apical ectodermal ridge (AER); however, not much is known about the molecular mechanisms underlying this regulation. SHH expression can be induced by the application of FGF‐soaked beads in the posterior region of the developing limb bud. DNA microarray analysis, following FGF bead application, revealed that LHX2 (LIM Homeobox 2) was upregulated >5‐fold. LHX2 encodes a transcription factor that is highly conserved across species, is expressed in the distal limb mesenchyme, and implicated in the integration of signaling inputs from the AER (FGFs), ZPA (SHH), and the dorsal ectoderm (WNT7a). Furthermore, LHX2/LHX9 double knockout mice show a marked reduction in SHH expression, confirming a role for LHX2 in regulating the signals that direct limb patterning and growth. Thus, we hypothesize that LHX2 is an intermediate in the FGF‐to‐SHH pathway. In order to test this hypothesis, we implanted FGF‐soaked heparin sulfate beads in embryonic chicken limb buds (Hamburger ‐ Hamilton stage 23), harvested the embryos, and assessed LHX2 expression in response to FGF induction via whole mount In situ hybridization (WMISH). Our results show that LHX2 is rapidly upregulated in response to FGF (<3 hrs). Its upregulation persisted through the experimental time course (24 hrs) and overlaps temporally with the upregulation of SHH in response to FGF. We therefore conclude that LHX2 is a downstream target of FGF and the early induction of LHX2 suggests that its regulation is direct. Further studies are underway to determine whether LHX2 can upregulate SHH and establish LHX2’s role as an intermediate in the FGF to SHH pathway. Grant Funding Source: Institutional