Msx2 Marks Early Craniofacial Mesenchymal Cells Preceding Osterix-Expressing Osteoblast Precursors

Objectives: A majority of craniofacial bones are formed through intramembranous ossification, in which mesenchymal cells directly differentiate into osteoblasts. Despite their well-described origins, the characteristics of early craniofacial mesenchymal cells are poorly understood. Msx2 is a transcription factor regulating Runx2 and osterix (Osx) in vitro, and mutations in MSX2 are associated with craniosynostosis and parietal foramina in humans. We hypothesize that Msx2 is expressed in early mesenchymal cells preceding Osx+ osteoblast precursors.
Methods: We conducted lineage-tracing studies to reveal the fates of Msx2+ cells in mice. We generated Msx2-creER transgenic lines to induce recombination transiently at various embryonic days upon tamoxifen administration. Recombination in a Rosa26-tdTomato reporter results in permanent labeling of Msx2+ cells, therefore their descendants can be followed over a chase period. An Osx-creER line was used to demonstrate the contrast with the fates of Osx+ osteoblast precursors.
Results: Msx2+ cells were found in the mandibular process of the first pharyngeal arch and the posterior cranial vault premordium at 24 hours after tamoxifen injection at embryonic day 9.5 (E9.5). After 6 days of chase at E15.5, their descendants became chondrocytes and perichondrial cells of the Meckel’s cartilage as well as osteoblasts and other mesenchymal cells in the mandible and the posterior calvaria. Postnatally, their descendants remained as dental pulp cells, odontoblasts and calvarial osteoblasts, while mostly disappearing from mandibular bones. Msx2+ cells marked after E11.5 demonstrated significantly less contribution to these mesenchymal cells. In contrast, Osx+ cells appeared only after E11.5, and their descendants were limited to but persistently provided osteoblasts well into postnatal days.
Conclusions: Msx2 marks early craniofacial mesenchymal cells preceding Osx expression, which are characterized with broader differentiation potentials into osteoblasts, chondrocytes and other mesenchymal cells. Transient nature of Msx2+ descendants among mandibular osteoblasts highlights the variable source of osteoblast precursors in the developing mandible.