Summary Msx 2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault

How migratory mesenchymal cells produce patterned structures is a key question in animal development. A compelling example of such a patterning process is the development of the mammalian skull vault. Comprising the frontal, parietal and squamosal bones, the skull vault develops from mesenchyme of neural crest and mesodermal origin (Couly et al., 1993; Jiang et al., 2002). These cells migrate to sites overlying the cerebral hemispheres where, in response to signals from the underlying neural tissue (Schowing, 1968), they coalesce, proliferate and differentiate along an osteogenic pathway. In later stages, the bones of the skull vault are united by sutures, fibrous joints that serve as growth centers and allow the skull vault to grow in concert with the brain (Wilkie, 1997; Cohen and MacLean, 2000; Wilkie and Morriss-Kay, 2001). The morphogenesis of the skull vault thus occurs in two phases, the first comprising the genesis, migration and initial specification of skeletogenic mesenchymal precursor cells, the second the differentiation of the skeletogenic mesenchyme and the ensuing appositional growth of the bones. The development of the skull vault is of interest not only because it provides a model of how mesenchymal populations produce patterned structures, but also because of its relevance to human disease (Wilkie, 1997; Cohen and MacLean, 2000; Wilkie and Morriss-Kay, 2001). Anomalies in skull vault development are common in humans, occurring as frequently as 1 per 2500 live births (Cohen and MacLean, 2000). Among these are craniosynostosis and persistent calvarial foramina. Craniosynostosis is the premature fusion of the calvarial bones at the sutures. Persistent calvarial foramina are defects in the ossification of bones of the skull vault. Several genes responsible for one or both of these defects have been identified (Wilkie, 1997; Wilkie and Morriss-Kay, 2001; Ornitz and Marie, 2002). These include FGF receptors 1, 2 and 3 (Jabs et al., 1994; Muenke et al., 1994; Reardon et al., 1994; Meyers et al., 1995), the basic HLH gene, Twist(Wilkie, 1997), and the homeobox genes M x2and Alx4(Wilkie and The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.