A Limbless Phenotype Uncovers an Essential Role for Shh in Amphibian Limb Initiation

Sonic Hedgehog (SHH) is a fundamental zone of polarizing activity (ZPA) morphogen well-characterized for a key role in vertebrate limb patterning. However, its function in amphibian limb development remains incompletely understood. By investigating the allotetraploid genome of Xenopus laevis , we demonstrate the functional and regulatory divergence between the two shh homeologs (L and S). Live imaging of locus-targeted reporters reveals that only shh . L with the evolutionarily conserved limb enhancer is expressed in the ZPA. Crucially, CRISPR-Cas9 disruption of shh . L shows its role extends beyond distal patterning: its ablation results in the complete absence of limbs. This limbless phenotype identifies the previously unknown shh function in limb formation from the initiation stage, which may be widely conserved across tetrapods.