73 - An ETS Binding Site Is Required for Full Activation of an LHX2-associated subAER Regulatory Module

Poster Board Number: 73
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Abstract Body : During limb development, proximodistal outgrowth is coordinated by fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER). Anteroposterior patterning and expansion is directed by the morphogen sonic hedgehog (SHH) secreted from the zone of polarizing activity (ZPA). Both SHH and FGF regulate each other’s expression in a positive reciprocal loop; however, the molecular intermediates involved are only partially characterized. The transcription factor LIM homeobox 2 (LHX2) is expressed in the distal limb mesoderm sub-adjacent to the AER and mediates the regulation of SHH by FGF. We have identified two LHX2-associated subAER regulatory modules (LASARM1 and LASARM2) active in the distal limb bud. Comparative in silico analysis of the DNA sequences of both LASARM1 and 2 reveals a 15 base pair DNA sequence conserved across vertebrate species that possesses a putative ETS transcription factor binding site which can mediate FGF signaling. Previously, we have shown that this putative ETS binding site is necessary for LASARM2 activity. Therefore, we hypothesized that this ETS binding site is necessary for LASARM1 activity.

To determine whether this ETS binding site contributes to LASARM1 activity, we performed single base-pair mutations of the ETS binding site sequence within a LASARM1-reporter construct followed by targeted regional electroporation into the distal limb bud mesoderm. Transfection efficiency was determined by co-transfection with a constitutive promoter-driven RFP expression vector. LASARM1 activity was determined by fluorescence microscopy 24 hours post transfection.

We found that mutation of the ETS binding site within LASARM1 resulted in a reduction of fluorescence (n=18) when compared to control. Our findings suggest that this putative ETS binding site contributes to LASARM1 activity. Moreover, these data provide a mechanism by which FGF, through ETS transcription factors activating LASARM1 or LASARM2, can regulate LHX2 expression. Further work is needed to confirm this connection.