Throughout its complex morphogenesis, the vertebrate skull must at once protect 
the brain and expand to accommodate its growth. A key structural adaptation that 
allows this dual role is the separation of the bony plates of the skull with 
sutures, fibrous joints that serve as growth centers and allow the calvarial 
bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of 
one or more calvarial bones with consequent abnormalities in skull shape, is a 
common developmental anomaly that disrupts this process. We found previously 
that a single amino acid substitution in the homeodomain of the human MSX2 gene 
is associated with the autosomal dominant disorder craniosynostosis, Boston 
type. This mutation enhances the affinity of Msx2 for its target sequence, 
suggesting that the mutation acts by a dominant positive mechanism. Consistent 
with this prediction, we showed that general overexpression of Msx2 under the 
control of the broadly expressed CMV promoter causes the calvarial bones to 
invade the sagittal suture. Here we use tissue-specific overexpression of Msx2 
within the calvarial sutures to address the developmental mechanisms of 
craniosynostosis and skull morphogenesis. We demonstrate that a segment of the 
Msx2 promoter directs reporter gene expression to subsets of cells within the 
sutures. In late embryonic and neonatal stages, this promoter is expressed in 
undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter 
activity is reduced in the suture, exhibiting a punctate pattern in 
undifferentiated osteoblastic cells in the outer margin of the osteogenic front. 
Overexpression of Msx2 under the control of this promoter is sufficient to 
enhance parietal bone growth into the sagittal suture by P6. This phenotype is 
preceded by an increase in both the number and the BrdU labeling of osteoblastic 
cells in the osteogenic fronts of the calvarial bones. These findings suggest 
that an important early event in MSX2-mediated craniosynostosis in humans is a 
transient retardation of osteogenic cell differentiation in the suture and a 
consequent increase in the pool of osteogenic cells.