ETS Binding Sites are 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 proximal-distal patterning, while sonic hedgehog (SHH) from the zone of polarizing activity (ZPA) directs anterior-posterior growth of the limb. These signaling centers maintain each other's expression in a reciprocal feedback loop; however, the molecular intermediates involved are only partially characterized. LIM homeobox 2 (LHX2) has been identified as an intermediate regulator downstream of FGF in the reciprocal loop. We have identified a cis-regulatory module (CRM) located upstream of the LHX2 promoter that is active in the sub-AER LHX2 expression domain. Overexpression of FGF in Hamburger-Hamilton stage 24 (HH24) chicken limb buds, resulted in increased expression of several FGF-associated transcription factors, including E26 transformation-specific proteins (ETS). Members of the ETS family have been known to regulate the expression of SHH via an enhancer known as the Zone of Polarizing Activity Regulatory Sequence (ZRS). We wondered whether the regulation of LHX2 was mediated through this LHX2-associated sub-AER Regulatory Module 1 (LASARM1) via ETS transcription factors. In silico analysis of LASARM1 revealed three ETS binding sites within LASARM1 and hypothesized that mutation of all three binding sites would disrupt FGF-mediated enhancer activity. To determine whether these ETS transcription factor binding sites contribute to LASARM1 activity, we performed site directed mutagenesis on all three ETS binding sites (triple∆ETS) within a LASARM1-ptk-eGFP reporter construct, followed by targeted regional electroporation (TREP) into the limb buds of HH24 chick embryos. 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. We found that mutation of all three ETS binding sites within LASARM1 resulted in fluorescence that was undetectable, suggesting reduced LASARM1 activity. Our findings suggest that FGF signaling utilizes ETS transcription factors to regulate LHX2 expression through the LASARM1 enhancer. Further work is needed to confirm this connection. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF-SHH regulatory loop.