Duplicated shh genes in Xenopus laevis reveal an essential role in early limb development

Sonic Hedgehog (SHH) is a key signaling molecule controlling anterior-posterior patterning in vertebrate paired appendages, but its requirement for initial appendage outgrowth varies across lineages. Whether SHH is genetically required for early limb-bud outgrowth in amphibians has remained unresolved due to embryonic pleiotropy caused by systemic SHH disruption. Here, we exploited the duplicated homeologs (shh.L and shh.S) in the allotetraploid frog Xenopus laevis to bypass systemic pleiotropy and investigate the limb-specific function of shh. We found that the conserved limb enhancer MFCS1/ZRS is retained at the shh.L locus but degraded at shh.S, resulting in limb-bud expression strictly confined to shh.L. Individual CRISPR/Cas9 disruption of shh.L, but not shh.S, led to a complete failure of initial limb-bud outgrowth and the absence of appendicular skeletal elements, without causing severe systemic defects. Our findings provide direct genetic evidence that shh.L is essential for initial limb outgrowth in X. laevis, demonstrating that dependence on SHH for early appendage initiation is strongly conserved in teleosts and amphibians but was reduced during amniote evolution.