Summary and conclusions

These results suggest that maintenance of proliferating osteogenic stem cells at the margins of the membrane bones that form the coronal suture (and on the inner and outer surfaces of these bones) depends on FGF levels being relatively low; higher levels of FGF are associated with osteogenic differentiation. The mitogenic signal is mediated by FGFR2 (and FGFR3). Higher levels of FGF2-stimulated FGFR2 signalling lead to down-regulation of Fgfr2 (and Fgfr3) gene expression and up-regulation of Fgfrl. Signalling through FGFR1 does not have a mitogenic outcome in this context, but leads to the expression of osteogenic differentiation genes. Once differentiation is well established, Fgfrl is down-regulated.

In the normal suture, this mechanism, involving differential levels of FGF from high in the differentiated region to low in the suture, ensures that sutural stem cell populations are maintained at the periphery of the growing bones. However, when receptor activation is increased, either experimentally (by the addition of exogenous FGF), or pathologically (by altered receptor function), FGFR2 is prematurely down-regulated and proliferation ceases. These conclusions are supported by the observation that FGF2 levels are increased within sutures at the time of fusion (Mehrara et al 1998). Also, in the synostosed coronal sutures of patients with

FIG. 5. The effect of exogenous FGF2 on gene expression in the region of the coronal suture: whole-specimen in situ hybridization on embryonic day (E) 16 mouse heads 24 hours after subcutaneous insertion of FGF2-soaked beads onto the coronal suture on E15. (A) Up-regulation of Fgfrl expression; (B) down-regulation of Fgfr2; (C) up-regulation of the bone differentiation marker, Osteopontin, in the region of the beads (see Iseki et al 1999, for details). Scale bar = 1 mm.

Crouzon syndrome, which is due to activating mutation of FGFR2 (Reardon et al 1994), FGFR2 immunoreactivity is reduced (Bresnick & Schendel 1995).

The role of FGFR3 signalling in development and maintenance of the coronal suture differs from that of FGFR2 in being involved in growth of both membrane and endochondral components of the skull: Fgfr3 is the only Fgfr gene expressed in cranial cartilage (Iseki et al 1999). However, the phenotypes of human FGFR3 mutations suggest that the skeletal growth response to FGFR3 signalling in endochondral bones of the skull and long bones are different. The achondroplasia phenotype (Gly380Arg mutation of FGFR3; Rousseau et al 1994, Shiang et al 1994) is characterized by decreased proliferation of epiphyseal chondrocytes, causing deficient growth of the long bones, but in the skull, growth is excessive, leading to macrocephaly. In thanatophoric dysplasia, a more severe phenotype resulting from mutation at different sites of the FGFR3 gene, the disparity between deficiency of long bone development and disproportionately increased head growth is even greater (see this volume: Wilkie et al 2000, Ornitz 2000, for details).

The hypothesis that Twist expression is essential for maintenance of osteogenic cell proliferation is supported by the observation that human TWIST mutations associated with craniosynostosis exert their effect via haploinsufficiency, in contrast to the gain-of-function mechanism of action of FGFR mutations. Twist expression is down-regulated during differentiation of cultured mouse calvarial osteoprogenitor cells, suggesting either that it promotes proliferation or that it inhibits differentiation (Murray et al 1992); it is known to inhibit differentiation during myogenesis (Hebrok et al 1994). If Twist is upstream of FGFR signalling in vertebrates, the overlap of the Twist and Fgfr2 expression domains in the coronal suture suggests that loss of function of Twist could lead to decreased osteogenic cell proliferation through failure to maintain adequate levels of Fgfr2 expression.

We suggest (1) that in the coronal suture, the rate of progression from proliferation to differentiation is maintained by interactions between TWISTregulated transcription, FGFR2/FGFR3 mitogenic signalling and FGFR1 differentiation-related signalling; (2) that differential levels of FGF2 (and possibly other FGF ligands) play a key role in these interactions through regional regulation of expression of the receptor genes.

A cknowledgements

We are grateful to F. Perrin-Schmitt, J. Heath and D. Ornitz for donation of probes. This study was funded by a Wellcome Clinical Training Fellowship award to D. Johnson, and an Action Research grant to G. M. Morriss-Kay.

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