(482.29) An E-Box Binding Site is Sufficient for ZPA Regulatory Sequence (ZRS) Activity in the Limb

Poster Board Number: C144
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

Kathryn Ball (Loma Linda University), Charmaine Pira (Loma Linda University), Kerby Oberg (Loma Linda University)

The zone of polarizing activity (ZPA) is a cluster of mesodermal cells that secrete Sonic hedgehog (Shh) to regulate anterior-posterior patterning during limb development. The ZPA regulatory sequence (ZRS) is the limb-specific enhancer that controls Shh expression. ZRS microdeletion abrogates Shh expression and posterior limb development, whereas ZRS single nucleotide variants (SNVs) produce anterior ectopic Shh and preaxial polydactyly. However, the ZRS mechanism of action is not well characterized. Transcription factors (TFs) Hand2, Twist1, and Hoxd13 regulate Shh expression and ZRS contains putative binding sites for each: two Hoxd13 binding sites and three E-boxes, sites targeted by basic helix-loop-helix (bHLH) TFs such as Hand2 and Twist1. Further, reports suggest these TFs may act through the ZRS: Hand2 purportedly binds the ZRS through a central E-box; as bHLH-TFs Hand2 and Twist1 may regulate ZRS as homo- or heterodimers; and both Hand2 and Hoxd13 activate the ZRS in vitro. Previously we demonstrated that disrupting all five binding sites eliminates ZRS activity. In this report, we further investigate these sites to determine the contribution of each to ZRS activity. We hypothesized that the Hand2 binding site is required for activity and that other E-boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm. We assessed ZRS activity via an enhancer-GFP reporter, altering all five binding sites to eliminate activity, then reinstating each site individually using site-directed mutagenesis. Constructs were electroporated into the presumptive limbs of chicken embryos (Hamburger-Hamilton stage 14). Enhancer activity was evaluated 48 hours post-electroporation by fluorescence microscopy. Restoring either Hoxd13 site led to weak, focal activity. Restoring two of the E-boxes had little to no effect, however, restoring the third, 3’ E-box was sufficient for robust activity. This was unexpected since the central E-box was reported as the likely Hand2 binding site. Our data suggest that the 3’ E-box is likely the critical site used by Hand2 in ZRS-mediated Shh regulation. Additionally, the Hoxd13 binding sites appear to impart focal activity supporting a role for localization. These findings further characterize the mechanism used to regulate focal Shh expression during limb development.

This research was funded in part by a grant from the LLU Pathology Research Endowment