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.
Authors
- Axel Visel (ORCID: https://orcid.org/0000-0002-4130-7784)
- Javier López-Rı́os (ORCID: https://orcid.org/0000-0001-6731-3798)
- Bárbara Pernaute (ORCID: https://orcid.org/0000-0002-8706-1126)
- Ana Alcaina‐Caro (ORCID: https://orcid.org/0000-0002-7352-5890)
- Gretel Nusspaumer (ORCID: https://orcid.org/0000-0002-3870-0964)
- Francisca Martinez Real (ORCID: https://orcid.org/0000-0003-0692-2260)
- Alicia Hurtado (ORCID: https://orcid.org/0000-0001-6267-704X)
- Darío G. Lupiáñez (ORCID: https://orcid.org/0000-0002-3165-036X)
- Ana Fernández‐Miñán (ORCID: https://orcid.org/0000-0002-9502-6785)
- Juan J. Tena (ORCID: https://orcid.org/0000-0001-8165-7984)
- Marco Osterwalder (ORCID: https://orcid.org/0000-0002-1969-2313)
- Holger Heyn (ORCID: https://orcid.org/0000-0002-3276-1889)
- Alejandro Díaz Rueda (ORCID: https://orcid.org/0009-0004-8252-0516)
- Ismael Sospedra-Arrufat (ORCID: https://orcid.org/0000-0001-6079-578X)
- Virginie Tissières (ORCID: https://orcid.org/0000-0003-4276-5514)
- Irepan Salvador‐Martínez (ORCID: https://orcid.org/0000-0002-3188-9784)
- Lucía Almorox (ORCID: https://orcid.org/0009-0008-1554-770X)
- Julia Joanna Pawluk (ORCID: https://orcid.org/0009-0000-0643-4043)
Institutions
- University of Bern (CH)
- Lawrence Berkeley National Laboratory (US)
- University Hospital of Bern (CH)
- Centro Andaluz de Biología del Desarrollo (ES)
- Centro Nacional de Análisis Genómico (ES)
Publication Details
- Journal
- bioRxiv (Cold Spring Harbor Laboratory)
- Published
- 2026-11-09
- DOI
- https://doi.org/10.64898/2026.11.07.765081
- Primary Topic
- Developmental Biology and Gene Regulation
- Type
- preprint