Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in 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 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated 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. Disruption of this pathway may lead to craniosynostosis.     

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.