Network inference at single-cell resolution reveals morphoregulatory divergence and multilayered buffering in limb evolution

How changes in conserved developmental genetic programs contribute to morphological evolution remains poorly understood, particularly in non-conventional model species. Here, we combine single-cell transcriptomic and chromatin-accessibility profiling with cross-species label transfer and gene regulatory network inference to compare mouse and pig limb development. Our analyses identify broadly conserved cellular organization but discrete divergence in HOX-, FGF- and SHH-regulated programs. FGFR inhibition in mouse limb buds phenocopies the distal expansion of pig GREM1 expression, while altered SHH responses and anterior-posterior polarity converge on the PTCH1 regulatory landscape. Its functional dissection reveals degeneration of a limb regulatory module in artiodactyls, conservation of an additional distal enhancer, and extensive redundancy among cis-regulatory inputs. Allele-specific analyses further show that reduced Ptch1 dosage is buffered by transcriptional compensation. Together, these findings reveal how cis-regulatory and pathway-level robustness can accommodate distributed regulatory change while preserving developmental function, providing a mechanistic framework for the evolution of artiodactyl limb morphology.