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

Alcaina-Caro, A.; Sospedra-Arrufat, I.; Tissieres, V.; Salvador-Martinez, I.; Almorox, L.; Pawluk, J. J.; Diaz-Rueda, A.; Fernandez-Minan, A.; Hurtado, A.; Martinez Real, F.; Lupianez, D.; Pernaute, B.; Visel, A.; Osterwalder, M.; Heyn, H.; Tena, J. J.; Nusspaumer, G.; Lopez-Rios, J.

doi:10.64898/2026.11.07.765081

bioRxiv · 10.64898/2026.11.07.765081

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

Alcaina-Caro, A.·Sospedra-Arrufat, I.·Tissieres, V.·Salvador-Martinez, I.·Almorox, L.·Pawluk, J. J.·Diaz-Rueda, A.·Fernandez-Minan, A.·Hurtado, A.·Martinez Real, F.·Lupianez, D.·Pernaute, B.·Visel, A.·Osterwalder, M.·Heyn, H.·Tena, J. J.·Nusspaumer, G.·Lopez-Rios, J.

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Abstract

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.

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Alcaina-Caro, A., Sospedra-Arrufat, I., Tissieres, V., Salvador-Martinez, I., Almorox, L., Pawluk, J. J., Diaz-Rueda, A., Fernandez-Minan, A., Hurtado, A., Martinez Real, F., Lupianez, D., Pernaute, B., Visel, A., Osterwalder, M., Heyn, H., Tena, J. J., Nusspaumer, G., Lopez-Rios, J.. 2026-11-09. Network inference at single-cell resolution reveals morphoregulatory divergence and multilayered buffering in limb evolution. https://doi.org/10.64898/2026.11.07.765081

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