Extracellular and cell membrane components of the FGFR signalling pathway

FGFR signalling requires the binding of two molecules of FGF to two receptors, resulting in homodimerization, trans-autophosphorylation of the tyrosine kinase domains, and signal transduction to the nucleus. The subsequent effects of the signal on gene transcription result in altered cell behaviour, involving either a change in the level of proliferation, presumably through an effect on cell cycle-related gene expression, or an effect on differentiation-related gene expression. The binding of FGF ligand to its receptor also requires the cooperation of a nonspecific receptor, heparin or a heparin-like molecule such as heparan sulfate proteoglycan (HSPG) (Yayon et al 1991). The activated dimeric receptor complex consists of a sandwich of 2xFGFR—heparin—FGF (Huhtala et al 1999). We have used immunohistochemistry of FGF2 ligand and an HSPG commonly found in developing tissues (perlecan), to discover the availability of these molecules for binding to FGFRs in the region of the coronal suture.

The immunohistochemical localization of FGF2 and perlecan in the E16 coronal suture are illustrated in Fig. 2B, C. Perlecan is present throughout the skeletogenic membrane, both within the suture and in the region of differentiated bone. This molecule is therefore a good candidate for involvement in FGF binding to FGFR throughout the growing skull vault, although it is likely that other HSPGs also play important roles, since disruption of the gene encoding perlecan has more severe effects on long bones than on the skull vault (Arikawa-Hirasawa et al 1999). Since the signals from the brain to the skeletogenic membrane may also be FGF ligands, it is interesting to observe that perlecan is also present in the meningeal layers between the brain and the skeletogenic membrane.

In contrast to perlecan, the distribution of FGF2 is much more specific: following secretion from the differentiating osteoblasts, it is present at high levels

FIG. 2. The mouse coronal suture. (A, B) Plane and organization of the sections shown in subsequent figures; the boxed area in (B) indicates the region illustrated. (C) Immunoreactivity of HSPG (perlecan) in the skin, skeletogenic membrane (frontal, parietal and coronal suture region) and meningeal tissue. (D) Immunoreactivity of secreted FGF2 in the region of the coronal suture: high levels are attached to osteoid and low levels diffuse into the extracellular matrix of the sutural mesenchyme and around the cells above and below the osteoid plates. c, cartilage; f, frontal bone domain; m, meningeal tissue; p, parietal bone domain. Scale bar = 100 ^m.

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attached to the osteoid (unmineralized bone matrix) of the differentiated region of the frontal and parietal bones, but at low levels in the extracellular matrix around the mesenchymal cells of the suture. This distribution suggests that there is a gradient (or possibly a stepwise change in concentration) of FGF from the region of differentiation to the region of proliferation, both within the suture, where proliferation leads to increase in size of the calvarial bones, and on the outer (skin side) and inner (brain side) surfaces of the bony plates, where proliferation allows increase in thickness of the bones. It is interesting to note that an FGF gradient has been reported to play a key developmental role in quite a different context, that of anteroposterior patterning (the determination of head, trunk and tail structures) in amphibian embryos, in which high levels of FGF—FGFR signalling are required for the specification of tail structures and low levels for head structures (Christen & Slack 1999).

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