(482.30) LHX2 Regulates Shh Expression in the Limb Independent of the ZPA Regulatory Sequence

Poster Board Number: C145
Introduction: AAA has separate poster presentation times for odd and even posters.
Odd poster #s – 10:15 am – 11:15 am
Even poster #s – 11:15 am – 12:15 pm

Kristen Whitley (Walla Walla University), Jessica Britton (Loma Linda University), Charmaine Pira (Loma Linda University), Kerby Oberg (Loma Linda University)

During limb development, proximal-distal patterning is controlled by fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER), while anterior-posterior expansion is coordinated by sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA). Both FGFs and SHH regulate each other through an autoregulatory loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate of the Fgf-to-Shh portion of the loop. In the absence of Lhx2 (and its redundant family member, Lhx9), Shh expression is markedly reduced. However, the mechanism by which LHX2 mediates FGF-regulation of Shh is unclear. Shh is regulated by a limb-specific enhancer called the ZPA Regulatory Sequence (ZRS). Using in silico analysis, our lab has identified two putative LHX2 binding sites within the ZRS. We hypothesized that LHX2 regulates Shh expression directly via the ZRS. To determine if LHX2 binding sites were necessary for ZRS activity, we mutated both LHX2 binding sites within a control tk-ZRS-eGFP reporter construct using site-directed mutagenesis. We electroporated these constructs into the presumptive limb buds of chicken embryos (Hamburger-Hamilton stage 14) and assessed for changes in ZRS activity in the limb buds 48 hours post transfection via fluorescence microscopy. We found that the mutation of both LHX2 binding sites within the ZRS resulted in activity similar to our control. These results suggest that LHX2 may not bind directly to the ZRS in its regulation of Shh. Instead, LHX2 may use another pathway to regulate Shh expression. Further experiments are needed to confirm this connection. Investigating the mechanism by which LHX2 regulates Shh will provide insight into how LHX2 contributes to coordinated patterned-limb development.