Differential gene expression between the proximal and distal developing calcaneus: Implications for primate evolution

Calcaneus morphological changes are important to our understanding of human and non-human primate locomotor evolution, including increased size and cross-sectional area of the calcaneal tuberosity in humans and distal end elongation in tarsiers and galagos. The calcaneus is the only tarsal to form a growth plate and associated secondary ossification center, which is located at the proximal end of the calcaneal tuberosity. Thus, distal elongation likely occurs independent of the growth plate and suggests at least two independent development modules define the calcaneus. While calcaneus morphology and evolution is frequently discussed, little is known about the genes underlying the development and morphology of this bone and its single growth plate. To address this deficiency, we used RNA sequencing to determine differentially expressed genes (DEGs) between the proximal (growth plate forming) and distal (non-growth plate forming) ends of the calcaneus in 0, 4, and 9 day old mice. On the proximal end, 27 DEG were expressed in at least 2 of the 3 ages. Gene ontology (GO) analysis found these to be associated with embryonic development and the retinoic acid signaling pathway. For the distal end, 148 DEGs were similarly identified and were surprisingly associated with functions related to muscle devel- opment, ion channels, and actin binding. However, genes known to affect skeletal development including Sall1 and Hoxa13 were also differentially expressed. This analysis identifies potential genetic targets with downstream morphological effects that can be targeted by natural selection to produce region-specific evolutionary changes.