96 - FGF Activates an LHX2-associated Cis-regulatory Module to Upregulate LHX2 Expression During Limb Development

Poster Board Number: 96
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Abstract Body : Proximodistal patterning in limb development is coordinated by fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER), while sonic hedgehog (SHH) directs anteroposterior patterning from the zone of polarizing activity (ZPA). The transcription factor LIM homeobox 2 (LHX2) is expressed in the distal mesoderm sub-adjacent to the AER. LHX2 is needed for FGF regulation of SHH; however, the mechanisms by which FGFs regulate LHX2 are unclear. We have identified several potential cis-regulatory modules (pCRMs), upstream of the LHX2 promoter, that contain transcription factor binding sites known to be targets of FGF signaling. We believe that FGF regulates LHX2 expression directly through a CRM. Therefore, we hypothesized that FGF works through one or more of these pCRMs to regulate LHX2 expression.

To determine whether these pCRMs are active, pCRM-GFP reporter constructs were transfected into the distal sub-AER chicken limb bud mesoderm when LHX2 expression is robust (Hamburger-Hamilton stage 23). Enhancer activity was assessed 24 hours after transfection via fluorescence microscopy. Since FGF2-soaked beads implanted into the proximal region of the limb bud can upregulate LHX2 expression, we evaluated whether FGF could also upregulate enhancer activity transfected into this same region. After transfection and bead implantation, embryos were incubated for 24 hours. Activity was determined and co-upregulation of LHX2 expression was confirmed using in situ hybridization.

We identified a CRM that showed robust enhancer activity in the distal limb bud overlapping LHX2 expression. Application of FGF2 in the mid-proximal region of the limb, up-regulated activity (n=6). These findings suggest that FGF upregulates LHX2 expression through a CRM to maintain SHH expression within the ZPA.