Morphological changes in calcanei of mice with Hoxa11 and Hoxd11 loss-of-function mutations

Hox11 genes are expressed around the developing wrist and ankle and are known to substantially impact pisiform shape and length in mice. The calcaneus is a tarsal bone that is paralogous to the pisiform in the wrist, but previous descriptions of mice with Hox11 mutations have suggested that little morphological change takes place unless Hoxa11 and Hoxd11 are both knocked out, at which point the calcaneus fails to form. However, these studies primarily relied on cleared and stained whole-mount specimens which limit resolution of morphological features. This study seeks to determine if calcaneus morphology is altered by three or fewer loss-of-function Hoxa11 and Hoxd11 alleles. We obtained microCT scans of 8 week old mice and compared calcaneus morphology in wild type mice and mice with one, two, and three Hoxa11 and Hoxd11 loss-of-function alleles. We used auto3dgm to conduct a 3D geometric morphometric analysis of shape variation using surface semi-landmarks. Principle components (PC) analysis indicates that calcaneus morphology is altered in mice with Hoxa11 and Hoxd11 loss-of-function mutations. PC1 accounts for 35.4% of shape variation and results from changes to the width and height of the calcaneal neck and shape of peroneal tubercle/process. PC2 accounts for 11.9% of shape variation and results from changes to the width of the calcaneal tuberosity and height of the posterior talar facet. Most specimens with either combination of three out of four Hoxa11 and Hoxd11 loss-of-function alleles cluster together. The other genotypes form a gradient of morphological change with WT, Hoxd11 heterozygotes, and Hoxd11 knockouts being most similar to each other and Hoxa11 heterozygotes, Hoxa11 knockouts, and heterozygotes for both genes being most similar to each other. These results suggest that Hox11 loss-of-function mutations result in altered calcaneus morphology and Hoxa11 and Hoxd11 loss-of-function mutations alter the shape of the calcaneus in different ways when fewer than three alleles are knocked out