Osterix is required for cranial neural crest-derived craniofacial bone formation

Abstract

Osx plays essential roles in regulating osteoblast and chondrocyte differentiation, and bone formation during mouse skeletal development. However, many questions remain regarding the requirement for Osx in different cell lineages. In this study, we asked whether Osx is required for craniofacial bone formation derived from cranial neural crest (CNC) cells. The Osx gene was conditionally inactivated in CNC-derived cells using a Wnt1-Cre recombination system. Neural crest-specific inactivation of Osx resulted in the complete absence of intramembranous skeletal elements derived from the CNC, and CNC-derived endochondral skeletal elements were also affected by Osx inactivation. Interestingly, Osx inactivated CNC-derived cells, which were recapitulated by lacZ expression, occupied the same regions of craniofacial skeletal elements as observed for controls. However, cells lost their osteogenic ability to differentiate into functional osteoblasts by Osx inactivation. These results suggest that Osx is important for craniofacial bone formation by CNC-derived cells. This finding provides novel insights of the regulation of craniofacial development by the gene network and transcription factors, and the understanding of human diseases caused by neural crest developmental abnormalities.

Fig. 1

Conditional inactivation of the Osx gene by Wnt1-Cre. Gross appearance (upper) and skeletal preparation (lower) of newborn mice. Skeletons were stained with alcian blue for cartilage followed by alizarin red for bone.

Fig. 2

Higher magnified lateral, dorsal, and basal images of the skeletons of Fig. 1. In Osx^flox/−; Wnt1-Cre mutants, the morphology of overall cartilage was conserved, but frontal and nasal bones, premaxilla, maxilla, and mandible were missing. Osx^flox/−; Wnt1-Cre mutants showed a progressively decreased size and mineralization in hyoid bone and rudimentary mandibles. n, nasal; f, frontal; p, parietal; ip, interparietal; s, supraoccipital; e, exooccipital; px, premaxilla; x, maxilla; md, mandible; tr, tympanic ring; z, zygomatic; bo, basioccipital; bs, basisphenoid; ps, presphenoid.

Fig. 3

Localization of lacZ expressing cells in Osx^flox/−; Wnt1-Cre mutants. LacZ expressing cells were stained blue by whole mount X-gal staining (A, upper) and mineral deposition was detected by von Kossa staining in X-gal stained sections (A, lower and B). In Osx^flox/− controls, mineral deposition occurred within matrix in association with lacZ expressing cells. However, mineral deposition was totally lacking in Osx^flox/−; Wnt1-Cre mutants.

Fig. 4

Histological analysis of Osx^flox/−; Wnt1-Cre mutants at 15.5 dpc (A) and in newborns (B). H and E, alcian blue, and von Kossa staining were performed in coronal sections of Osx^flox/− controls and Osx^flox/−; Wnt1-Cre mutants. Alcian blue and nuclear fast red staining showed no change in cartilage formation in either group. However, von Kossa staining showed the complete absence of mineral deposition in Osx^flox/−; Wnt1-Cre mutants at 15.5 dpc and in newborns.