An ELK4 Binding Site is Critical for Activity of the LHX2‐Associated Sub‐AER Regulatory Module 1 (LASARM1)

Abstract

Fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) coordinate limb growth and proximal‐distal patterning, while sonic hedgehog (SHH) from the zone of polarizing activity directs anterior‐posterior growth and patterning. Moreover, these signaling centers maintain each other’s expression in a reciprocal feedback loop. This regulatory loop coordinates limb patterning during outgrowth. However, the molecular intermediates involved in in this loop are only partially characterized. Previously, we identified LIM homeobox 2 (LHX2), expressed in the mesoderm subjacent to the AER, as a downstream target of FGF in the FGF to SHH arm of the feedback loop.LHX2‐associated Cis‐regulatory modules (CRMs) are reasonable candidates to convey the FGF‐mediated regulation of LHX2. We have identified a 1745 bp CRM located 483 kb upstream of the LHX2 promoter that is active in the sub‐AER region within the expression domain of LHX2. In silico analysis of this LHX2‐associated sub‐AER regulatory module 1 (LASARM1) revealed several FGF‐related transcription factor binding sites, including a site for ETS like‐4 (ELK4). We performed in situ hybridization for ELK4 mRNA and found a pattern coincident with LHX2 expression. Thus, we hypothesized that mutation of the ELK4 binding site within LASARM1 would disrupt FGF‐mediated enhancer activity.To identify whether the putative ELK4 transcription factor binding site contributes to LASARM1 activity, we performed site‐directed mutagenesis on the ELK4 binding site in the LASARM1‐ptk‐EGFP reporter construct. We used targeted regional electroporation (TREP) to introduce control and mutated reporter constructs into the distal mesoderm of Hamburger‐Hamilton stage 23 chicken limb buds. Transfection efficiency was determined by co‐transfection with a β‐actin promoter‐driven RFP construct. After 24 hours of incubation LASARM1 activity was determined by fluorescence microscopy.The fluorescence of LASARM1mutELK4 was markedly reduced relative to the LASARM1control. Our findings suggest that FGF signaling utilizes the ELK4 transcription factor to regulate LHX2 expression through the LASARM1 enhancer. Further work is needed to confirm this connection.