Divergent expression and conserved function of Alx4 for limb development in amniotes

Abstract

Morphological diversification among amniote limbs is widely thought to arise through evolutionary changes in developmental gene regulation, yet whether divergent gene expression is accompanied by divergence in developmental function remains unclear. Here, we examined this question using Alx4 , an Aristaless-like homeobox gene required for vertebrate limb development. Comparative expression analysis showed that Alx4 exhibits divergent spatial expression patterns in chicken and mouse limb buds. In contrast, evolutionary sequence analysis across 51 amniote species revealed strong conservation of the coding sequence, including complete conservation of key functional domains and pervasive purifying selection. Cross-species gain-of-function assays in chicken embryos showed that chicken, mouse, and human Alx4 orthologs induce highly similar limb phenotypes, characterized mainly by skeletal shortening and less frequently by tibial widening, syndactyly, and ectopic sesamoid-like cartilage. Transcriptomic profiling further identified shared downstream responses involving extracellular matrix organization, focal adhesion, Wnt signaling, and skeletal development. These findings indicate that Alx4 retains a conserved developmental function despite divergence in limb bud expression, suggesting that differences in Alx4 regulation may contribute to variation in limb development, whereas changes in Alx4 protein function are less likely to explain the observed evolutionary differences.