Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme

Craniofacial bones derive from cephalic neural crest, by endochondral or intramembranous ossification. Here, we address the role of the homeobox transcription factor Dlx5 during the initial steps of calvaria membranous differentiation and we show that Dlx5 elicits Runx2 induction and full osteoblast differentiation in embryonic suture mesenchyme grown "in vitro". First, we compare Dlx5 expression to bone-related gene expression in the developing skull and mandibular bones. We classify genes into three groups related to consecutive steps of ossification. Secondly, we study Dlx5 activity in osteoblast precursors, by transfecting Dlx5 into skull mesenchyme dissected prior to the onset of either Dlx5 and Runx2 expression or osteogenesis. We find that Dlx5 does not modify the proliferation rate or the expression of suture markers in the immature calvaria cells. Rather, Dlx5 initiates a complete osteogenic differentiation in these early primary cells, by triggering Runx2, osteopontin, alkaline phosphatase, and other gene expression according to the sequential temporal sequence observed during skull osteogenesis "in vivo". Thirdly, we show that BMP signaling activates Dlx5, Runx2, and alkaline phosphatase in those primary cultures and that a dominant-negative Dlx factor interferes with the ability of the BMP pathway to activate Runx2 expression. Together, these data suggest a pivotal role of Dlx5 and related Dlx factors in the onset of differentiation of chick calvaria osteoblasts.