The ZPA Regulatory Sequence (ZRS) Hand2 and Twist1 Binding Sites are Not Necessary for ZRS Activity

Abstract

Anterior-posterior patterning in the developing limb is regulated by Sonic Hedgehog (Shh), which is expressed in the Zone of Polarizing Activity (ZPA). Shh is regulated by a limb-specific enhancer known as the ZPA Regulatory Sequence (ZRS). Mutations in the highly conserved ZRS have been linked to limb malformations in humans. Single nucleotide variants (SNVs) cause digit duplications, such as preaxial polydactyly (PPD) and triphalangeal thumb-polysyndactyly syndrome (TPTPS). The ZRS mechanism is not well understood. Conditional knockout of Hand2 results in a delta-Shh phenotype, indicating the importance of Hand2 in Shh regulation. Hand2 and Twist1 are basic helix-loop-helix (bHLH) transcription factors, capable of homo- and hetero-dimerization, that bind E-boxes (CANNTG) with some promiscuity. The objective of the present study was to determine whether the three ZRS E-boxes are necessary for activity. We hypothesize that altering E-boxes will disrupt ZRS function. Enhancer activity was evaluated using a ZRS-GFP reporter plasmid. We generated a construct with all three E-boxes altered using site-directed mutagenesis. Constructs were electroporated into presumptive forelimb buds in Hamburger-Hamilton stage 14 chicken embryos. Enhancer activity was recorded using fluorescent microscopy. Surprisingly, modification of the Hand2 and Twist1 binding sites did not affect ZRS activity. Future work is needed to identify what transcription factors are indispensable for ZRS regulation of Shh.