Molecular signaling in suture development

Abstract

The aim of this study (1) was to develop an in vitro organ culture system to facilitate the study of molecular signaling in the developing calvarial bones and sutures. Calvaria of mice aged 15 embryonic days (E15) were dissected free under a stereomicroscope using ophthalmic scissors. Explants were placed in a Trowell-type organ culture and observed for 1-4 days. The explants developed in a manner consistent with suture development in vivo. The opposing parietal bone osteogenic fronts (OFs) approximated but did not fuse. In vitro osteoblast activity, analyzed by in situ hybridization for bone sialoprotein, correlated well with in vivo development. In situ hybridization on both whole mount and sectioned tissue was carried out to detect mRNA for fibroblast growth factor receptors (Fgfr's), Fgf's and Msx's, genes known to cause human craniosynostosis or thought to be involved in calvarial development. To test whether growth factors interacted with each other, beads impregnated with the molecule under investigation were placed in various locations and their effects analyzed. Bek, a splicing alternative of Fgfr2, was intensely expressed in the OFs of parietal bones of E15 and E17 mouse embryos. Postnatally, its intensity diminished. Fgf2, a ligand known to bind to Fgfr2, was intensely expressed in the sutural mesenchyme between the OFs at 15 embryonic days; this had declined at 17 embryonic days, and transcripts were not detected postnatally. Msx1 was expressed in the sutural mesenchyme and the underlying dura mater during embryonic and postnatal stages. Embryonically, Msx2 was intensely expressed in locations similar to those of Msx1. However, after birth the expression of Msx2 was dramatically diminished in the mesenchyme, and it completely disappeared from the dura mater. Beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. In conclusion, we have developed microdissection techniques and an organ culture system for the developing mouse calvaria. We suggest that the local application of FGF on the osteogenic fronts, which accelerates suture closure, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors.