The Pattern of EGR1 Expression During Chick Limb Development Suggests a Role in Regulating SHH Expression and/or Termination

Limb development is governed by a complex interplay between three signaling centers that mediate growth along three coordinate axes. The apical ectodermal ridge (AER), zone of polarizing activity (ZPA) and the dorsal ectoderm regulate the proximal‐distal, anterior‐posterior and dorsal‐ventral axes, respectively. Development occurs along these three axes to create an asymmetrical limb and is highly dependent on crosstalk between fibroblast growth factors (FGFs) from the AER and sonic hedgehog (SHH) from the ZPA. The mechanisms and intermediate players between FGF and SHH are poorly characterized. Previous work in our lab identified a number of FGF‐responsive candidate genes. Most notably early growth response 1 (EGR1), encoding a zinc finger type protein, was identified as the gene with the most robust induction in response to FGF. EGR1 has been shown to play a role in tendon development but to date its expression in the developing limb has not been well characterized. We found EGR1 to have low endogenous expression in the chick limb by whole mount in situ hybridization (WMISH). We highlighted the WMISH pattern of EGR1 using a 3 hr cycloheximide pretreatment to enhance RNA accumulation. This approach revealed that EGR1 is expressed subjacent to the AER and overlaps the ZPA during robust SHH expression (Hamburger‐Hamilton stage 17 (HH17) through HH24). In contrast, as SHH expression wanes (HH25–26), EGR1 expression recedes from the posterior ZPA, and persists in the anterior progress zone underlying the AER. In addition, EGR1 expression extends proximally from the anterior border of the progress zone. The reduction of EGR1 within the ZPA coincident with the decline in SHH may indicate a role for EGR1 in optimizing SHH expression and triggering termination.