Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynos tosis, the former by constitutive activation and the latter by haploinsuffi ciency. Although clinically achieving the same end result, the premature fu sion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later i n suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is a pplied via beads to the osteogenic fronts, suture closure is accelerated (K im, H.-J., Rice, D, P. C,, Kettunen, P. J, and Thesleff, I. (1998) Developm ent 125, 1241-1251). In order to investigate further the role of FGF signal ling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fg fr4, as well as their potential ligand Fgf2, The IIIc splice variants of Fg fr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by different iating osteoblasts at the osteogenic fronts (E15), In comparison to Fgf9, F gf2 showed a more restricted expression pattern being primarily expressed i n the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6), Twist and Id1 wer e expressed by early preosteoblasts, in patterns that overlapped those of t he FGF ligands, but as these cells differentiated their expression dramatic ally decreased. Signalling pathways were further studied in vitro, in E15 m ouse calvarial explants, Beads soaked in FGF2 induced Twist and inhibited B sp, a marker of functioning osteoblasts, Meanwhile, BMP2 upregulated Id1, I d1 is a dominant negative HLH thought to inhibit basic HLH such as Twist, I n Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We d emonstrated that in Twist(+/-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disru ption of this pathway may lead to craniosynostosis.