Identifying growth plate specific genes using the unusual ossification of the mammalian metatarsal and pisiform

Primates vary in skeletal proportions, and humans have uniquely shorted pisiforms. Both are the result of regulation of growth plate patterning. Genes regulating differential growth are generally unknown. We utilize normal variation in mammalian skeletal ossification to identify genes specific to growth plate patterning. Third metatarsals (MT3) form only a single distally placedgrowth plate, and the pisiform (in non-humanmammals) is the only carpal to form a growth plate. We compared gene expression in distal versus proximal MT3s and pisiform versus other carpals; tissue pairs that control for the effects of age, systemic growth factors, and biomechanical environment. Using RNA-seq in 4- and 9-days old mice we identify numerous differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed MT3 DEGs are disproportionately associated with limb development/growth and factors associated with the Fzd8-Ror2-Wnt5a pathway. Significant pisiform-carpal GO terms include skeletal and cartilage development and abnormalities of the wrist. We identified a limited set of DEGs shared by both the MT3 and pisiform-carpal comparisons, including Wnt5a. Wnt5a is expressed in the perichondrium and columnar/hypertrophic cell boundary in both the distal and proximal MT3. However, the related Wnt10a gene is strongly expressed in the distal but not proximal MT3 bone collar. This conforms with previously identified roles for Wnt genes in regulating osteogenesis and cell polarity within the growth plate and provides potential genetic targets that may harbor regulatory adaptations associated with the evolution of primate limb proportions and human pisiform reduction.