During limb development a cluster of mesodermal cells in the distal posterior limb bud, the zone of polarizing activity (ZPA), regulates patterning along the anterior-posterior axis. The signaling molecule responsible for this patterning is Sonic hedgehog (Shh), which is controlled by a highly conserved limb-specific enhancer, the ZPA regulatory sequence (ZRS).ZRS microdeletion results in limbs that fail to express Shh and do not develop posterior limb elements, whereas many ZRS single nucleotide variants (SNVs) lead to anterior ectopic Shh expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. The ZRS contains putative binding sites for Hand2, Twist1, and Hoxd13: transcription factors (TFs) known to regulate Shh. Both Hand2 and Twist1 are basic helix-loop-helix (bHLH) TFs and are thought to function as homo- or heterodimers, while Hoxd13 has been shown to interact with Hand2.The present study's objective is to determine the role of these TFs in ZRS function. We hypothesize that altering Hand2, Twist1, and Hoxd13 bindings sites, either alone or in concert, will disrupt ZRS activity. To evaluate enhancer activity, ZRS was cloned into an enhancer-GFP reporter construct. Hand2, Twist1, and Hoxd13 binding sites were altered using site-directed mutagenesis, and constructs were electroporated into the presumptive limb buds of chicken embryos (Hamburger-Hamilton stage 14). GFP expression was evaluated 48 hours post-electroporation. We determined that mutating the binding sites for all three TFs in concert resulted in loss of ZRS activity. However, constructs with either Twist1 or Hoxd13 binding sites intact remained active. These data suggest that Twist1 and Hoxd13 may have either redundant or temporally distinct roles in ZRS regulation of Shh. Future work will determine whether these TFs bind the ZRS at the specific sites we have identified.