Loss of HAND2 and HOXD13 Binding Sites in the ZPA Regulatory Sequence (ZRS) is not Sufficient to Disrupt Activity in the Limb

Abstract

The ZPA regulatory sequence (ZRS) is a limb‐specific enhancer that localizes Sonic Hedgehog (SHH) expression to the zone of polarizing activity (ZPA), which consequently regulates anteroposterior patterning in the developing limb. ZRS deletion lacks SHH expression in the limb resulting in loss of posterior limb structures, while ZRS single nucleotide variants (SNVs) can lead to ectopic SHH expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. HAND2 and HOXD13 are key transcription factors in SHH regulation that bind to the ZRS, potentially modulating SHH transcription. The present study’s objective was to determine the role of HAND2 and HOXD13 binding sites in ZRS function. To evaluate ZRS enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. HAND2 and HOXD13 binding sites were altered using site‐directed mutagenesis, and contructs were electroporated into presumptive limb buds in Hamburger‐Hamilton stage 14 chicken embryos. GFP expression was evaluated 48 hours post‐electroporation. ZRS with mutated HAND2 and HOXD13 binding sites retained activity in the limb bud in a pattern that overlapped ZRS activity. This is unexpected since HAND2 and HOXD13 are known to be critical for SHH expression. Future work is needed to clarify the binding site(s) critical to activity and to characterize the factors involved in ZRS function.Support or Funding InformationSupported in part by a grant from the Department of Pathology, Loma Linda University.