Embryonic muscle activity redirects avian humeral shape development

The development of the tetrapod appendicular skeleton follows a conserved sequence of chondrogenesis and endochondral ossification, yet the evolutionary-developmental mechanisms that have yielded the immense morphological diversity of the vertebrate skeleton remain incompletely understood. Mechanical stress from embryonic muscle activity is a key regulator of skeletal development, and its absence can lead to several osteological abnormalities. Although the effects of altered embryonic movement on skeletal rudiment morphology are well documented, the relationship between experimentally induced developmental shape changes and large-scale evolutionary variation in skeletal form remains comparatively unexplored. Here, we assess the influence of embryonic muscle activity on skeletal shape by inducing paralysis in developing chicken embryos (Gallus gallus). We used microCT imaging and geometric morphometrics to quantify differences in humeral size and shape between paralyzed and control embryos and to compare these patterns with humeral morphology across extant bird diversity. We also examined the spatial distribution of proteins associated with endochondral ossification and cell proliferation. Paralysis did not affect overall humeral size but did cause marked changes in humeral curvature and the growth pattern of the distal epiphysis. Paralyzed and control humeri followed distinct developmental trajectories, with the most pronounced shape differences concentrated in the proximal epiphysis. Notably, paralyzed embryos fell outside the range of all sampled adult birds along PC3 of the proximal epiphysis. These findings suggest that embryonic muscle forces may contribute to evolutionary variation in avian limb shape.