AER‐Related FGFs Upregulate LHX2 Through MEK in the RAS‐Associated Signaling Pathway During the Maintenance of ZPA‐Related SHH Expression

Abstract

Fibroblast growth factors (FGFs) and sonic hedgehog (SHH) are mitogens known to affect limb growth and patterning during embryonic development. While FGFs establish the proximal‐distal axis from the apical ectodermal ridge (AER), SHH directs anterior‐posterior patterning from the zone of polarizing activity (ZPA). In addition, FGFs and SHH act in an autoregulatory loop by maintaining each other’s expression to coordinate limb patterning during limb outgrowth. The LIM Homeobox 2 (LHX2) transcription factor, which acts in the progress zone (PZ), was identified as an intermediary in the FGF to SHH arm of the maintenance loop. FGF mediates its action on cells through multiple intracellular signaling pathways including the JAK‐STAT self‐renewal pathway, PKC cell motility pathway, PDK1‐AKT cell survival pathway, and RAS‐associated cell proliferation pathway. We wondered whether one or more of these pathways directed FGF‐mediated upregulation of LHX2 in the FGF‐SHH regulatory loop. To evaluate a specific FGF signaling pathway, we implanted a bead soaked in a selective pathway‐specific inhibitor adjacent to an FGF2‐soaked bead in the posterior margin of Hamburger‐Hamilton stage 23–24 (HH23‐HH24) chicken limbs, a region known to induce LHX2 expression in response to FGF. The embryos were harvested after 4 hours and assayed for LHX2 and SHH expression by whole mount in situ hybridization. Ectopic LHX2 and SHH expression levels were reduced in the presence of the RAS and MEK inhibitors, indicating that FGF requires functional RAS‐associated pathways that utilize MEK to upregulate LHX2 expression.