Limb development occurs along three major axes – proximal‐distal, anterior‐posterior, and dorsal‐ventral. Fibroblast growth factors (FGFs) secreted by the apical ectodermal ridge (AER) and sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA) are responsible for proximal‐distal and anterior‐posterior development, respectively, and maintain each other through a positive feedback loop. This reciprocal loop is critical for proper limb development. Recently, we identified LIM homeobox 2 (LHX2) as an intermediate in FGF‐mediated SHH expression. There are over 25 conserved regions of non‐coding DNA associated with the LHX2 gene locus that could serve as regulatory modules and targets of FGF signaling. We hypothesize that FGF regulates LHX2 through at least one of these potential cis regulatory modules (PCRMs). We selected 10 PCRMs with active chromatin marks in the limb and screened for their activity within the LHX2 expression domain (distal mesoderm subjacent to the AER). Each PCRM was inserted into the pTK‐GFP reporter and electroporated into the distal mesoderm of HH20‐23 chicken embryo wing buds. PCRM activity was determined 24 hours later using fluorescence microscopy. We found three of the PCRMs display activity (CRM (−19), CRM (−2), and CRM (−1)) that overlap LHX2 expression in the chicken wing bud. One CRM (−19), approximately 130 kb upstream of the LHX2 locus, is most consistent with the pattern of LHX2. Further studies are underway to determine whether these CRMs interact with the LHX2 promoter and whether FGF regulates their activity.Support or Funding InformationFunded in part by a grant from the LLU Pathology Endowment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Limb outgrowth is controlled, in part, by fibroblast growth factor (FGF) and sonic hedgehog (Shh). FGF up‐regulation of SHH is essential for coordinated limb pattering; however, not much is known about this mechanism. We recently found that LHX2 is an intermediate in FGF‐mediated SHH up‐regulation. LHX2 is part of a family of genes that are known to integrate signaling events. Thus, we hypothesized that FGF would up‐regulate LHX2 through associated potential cis regulatory modules (PCRM). Multiple PCRMs were found in association with the LHX2 locus. PCRMs were screened using comparative genomics, proximity to the LHX2 promoter, and transcription factor binding sites related to FGF signaling. To exam the role of suspected PCRMs, we electroporated PCRM‐reporter constructs into chick embryos. We found one PCRM with activity in the brain coincident with LHX2 expression, but none of our initial PCRMs exhibited activity in the limbs. We reevaluated the LHX2‐associated PCRMs in light of limb‐specific chromatin activation marks and identified 5 additional PCRMs for analysis. Studies are underway to determine limb‐related PCRM activity of this new cohort. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF‐SHH regulatory loop.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.