INTRODUCTION AND OBJECTIVES: 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).

METHODS: 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.

RESULTS AND CONCLUSIONS: 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.



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