A conserved differential growth mechanism drives interdigital remodelling and digit individualization in birds

The autopod, the distal region of tetrapod limbs, represents a key evolutionary innovation requiring coordinated patterning, proliferation, and morphogenetic processes. While avian digit individualization has classically been described as driven almost exclusively by Programmed Cell Death (PCD) in the interdigital zone (IDZ), evidence from basal tetrapods indicates that differential growth between the digital zone (DZ) and IDZ originally mediated digit separation without apoptotic involvement. It remains unclear if this ancestral mechanism persists in amniotes or has been evolutionarily replaced by apoptosis. Here, we investigated growth dynamics in chicken (Gallus gallus) embryos by combining quantitative extension analyses, fluorescent DiI lineage tracing, and PH3-based proliferation assays. We demonstrate that differential growth is conserved in amniotes and arises through dynamic divergence in DZ and IDZ behaviour in chicken embryos. In chicken, although both regions expand comparably until HH32, IDZ extension declines markedly thereafter, while DZ outgrowth continues uninterrupted. DiI lineage tracing shows that the medial and proximal IDZ regions exhibit significantly greater extension at HH32 than at HH34, indicating a developmental decrease in growth that does not occur in the DZ, which maintains higher, stable extension rates across stages. Proliferation analyses align with these morphometric findings: the IDZ shows a sharp reduction in PH3-positive cells post-HH32, establishing a proliferative deficit relative to the DZ. Examination along the proximodistal axis revealed that the distal IDZ, influenced by the AER, shows the greatest proliferative activity, whereas medial and proximal regions contain fewer proliferative cells; this gradient is absent in the DZ, which displays relatively uniform proliferation. Together, these results indicate that avian digit separation is not governed solely by apoptosis but is initiated by an intrinsic decline in IDZ extension and cell-cycle activity. We propose that birds retain the ancestral differential growth mechanism, which works in concert with apoptosis to refine digit boundaries, bridging the evolutionary bridge between amphibian-like growth-driven individualization and amniote apoptotic refinement.