Mesenchymal Regulation of Osteogenic Differentiation During Development

INTRODUCTION With the goal of devising new therapies for disease, birth defects, and injuries to the skeleton, there is keen interest in understanding how mesenchymal cells differentiate into osteocytes and ultimately make bone. A wide range of in vitro and in vivo studies have revealed that osteogenesis is a complex process involving numerous gene regulatory networks, reciprocal signaling interactions, and multiple hierarchical levels of control. Yet what remains unclear are precise mechanisms that initiate and synchronize the progression of mesenchyme through each discrete step of osteogenesis including induction, proliferation, differentiation, osteoid deposition, and mineralization. This is significant since a major clinical objective is to engineer mesenchyme for in vivo applications. To address this issue, we take a systems-level strategy and experimentally manipulate neural crest mesenchyme (NCM) destined to form all the bone in the jaw skeleton. We have established a unique avian chimeric transplant system that exploits the divergent maturation rates of quail and duck. By transplanting faster-maturing quail NCM into slower-developing duck (“quck”) or conversely, duck NCM into quail (“duail”), we can assess the extent to which each step of osteogenesis is directed by donor NCM, and more importantly, we can uncover and dissect apart potential mechanisms. To do so, we assay for donor-induced changes to the timing of histological events and screen for candidate genes that become differentially expressed in quck. We then use gainand loss-of-function approaches to test if these genes account for an observed phenotype. Previously we have shown that osteogenesis is significantly accelerated in chimeric quck and delayed in duail. In both cases, NCM shifts entire programs for the induction, differentiation, and mineralization of bone. How NCM accomplishes such a complex task, and what factors are sufficient to replicate this phenomenon is unknown. We hypothesized that one likely candidate would be Runx2, since its expression was altered in chimeras. In vitro, Runx2 can direct mesenchymal cells down the osteoblast lineage, and trigger expression of genes encoding major protein components of bone matrix, including Col1. Moreover, binding of Runx2 to ribosomal DNA leads to direct repression of ribosomal RNA synthesis to help modulate the switch from proliferation to differentiation. Similarly, our experiments show that Runx2 over-expression strongly enhances differentiation and mineralization in both primary chick mesenchymal cell cultures and a chick fibroblast cell line. In contrast to such in vitro data, we find that in vivo over-expression of Runx2 is not sufficient to alter the timing of either differentiation or mineralization. Thus, NCM must employ additional molecular mechanisms to control osteogenesis.