Characterization of Hox gene expression during fracture repair and the functional characterization of Mustn1 during development and chondrocyte differentiation

Previously, our laboratory identified several differentially regulated genes during bone fracture repair (BFR). Of these genes, several from the Homeobox family known to be active during development were spatially and temporally localized during BFR. That their activation during BFR is consistent with their roles in development adds support to the idea that development and BFR are analogous processes. Further research was preformed on the musculoskeletal temporally activated novel gene (Mustang a.k.a. Mustn1). This small (9.6kDa) nuclear protein was found to be strongly up-regulated during the early stages of bone fracture repair, especially in the periosteum, osteoblasts, and proliferating chondrocytes. Mustn1 expression was also found to be specific to the musculoskeletal system in adult vertebrates. Further characterization of Mustn1 revealed that this gene is highly expressed in areas of active chondrogenesis, specifically during development of the limb buds, branchial arches, somites and posterior tail. This localization pattern strongly suggests that this gene plays a role during musculoskeletal development in mouse. To further study the role of Mustn1, we functionally perturbed its expression in the pre-chondrocyte cell line RCJ3.1C5.18 (RCJ) via stable overexpression and RNAi silencing. While overexpressing Mustn1 (at ∼2-6 fold levels) in RCJ cells had no effect on chondrocyte proliferation or differentiation, downregulating Mustn1by 52-66% resulted in significant repression of both proliferation rate and matrix/proteoglycan production. These effects were also accompanied by the downregulation of the chondrogenic differentiation markers, Sox9, Collagen II, and Collagen X. Moreover, these inhibitory effects were rescued when Mustn1 was reintroduced into the silenced cell line. To further elucidate its role in vivo, we suppressed Mustn1 production in Xenopus laevis embryos via antisense morpholino injection. Following Mustn1 suppression, embryos displayed gross craniofacial and musculoskeletal defects, e.g. small, or loss of, eye(s), shortened body axis, as well as kinks within the tail, indicative of disturbances in cartilage and skeletal muscle formation. These defects were reduced in severity or completely ablated upon reintroduction of Mustn1 mRNA into Mustn1 morpholino injected embryos. In addition, the expression of the myogenic differentiation marker MyoD was not altered. However, when Sox9 was assayed in the same manner, its expression was reduced by ∼40% in Mustn1 morpholino injected embryos when compared to the control embryos especially within the branchial arches, neural crest cells, and anterior craniofacial regions. Again, reintroduction of Mustn1 RNA rescued the expression pattern of Sox9 in Xenopus embryos. These data strongly suggest that Mustn1 is a critical player in chondrogenesis during vertebrate development.