BoneKEy-Osteovision | Commentary

FGF signaling in calvarial bone development



DOI:10.1138/2002043

Commentary on: Moore R, Ferretti P, Copp A, Thorogood P. Blocking endogenous FGF-2 activity prevents cranial osteogenesis. Dev Biol. 2002 Mar 1;243(1):99-114.

The bones of the cranial vault are, in the main, formed by intramembranous ossification. At birth the sutures separating these bones are patent which allows for continued postnatal development and growth of the skull. The brain also continues to develop postnatally, and if its expansion is restricted by the premature fusion of calvarial sutures skull growth may be redirected to accommodate this. This abnormal condition called craniosynostosis can lead to a change in skull shape and deformity. Mutations in fibroblast growth factor receptors 1, 2 and 3 (FGFR1, 2 and 3) (OMIM 136350, OMIM 176943, 134934) are known to cause both syndromic and non-syndromic forms of craniosynostosis. There is evidence these mutations cause craniosynostosis by ligand independent constitutive activation. The mutation may cause reduced ligand dissociation, altered covalent cross linking and transmembrane hydrogen bonding, increased affinity for FGF ligands, receptor isoform switching, and increased calvarial cell differentiation and bone matrix formation ().

Several genetically engineered mouse models affecting Fgfrs have been generated. Knocking out the Fgfr1 gene as well as deleting the third immunoglobulin-like loop of Fgfr2 have been of little use in studying craniosynostosis pathogenesis as they both result in death before the start of skeletal development. However, mice carrying the Pfeiffer syndrome mutation (Pro250Arg substitution) in Fgfr1 exhibit premature fusion of calvarial sutures (). The Bulgy-eye or Bey mouse has been generated by retroviral insertional mutagenesis in embryonic stem cells. The vector is inserted between the Fgf3/Fgf4 locus causing an upregulation of both Fgf3 and Fgf4. Heterozygous Bey mice exhibit facial shortening and premature closure of several calvarial sutures (). Mice hemizygous for Fgfr2c exhibit coronal synostosis. In addition to this calvarial phenotype the bones making up the lower orbital rim fuse early leading to shallow orbits and proptosis, a feature common to both Apert and Pfeiffer syndromes (). At first sight this phenotype would seem to contradict the gain of function mutations found in syndromic craniosynostosis patients. However, the partial loss of Fgfr2c confers a gain of function in the alternative splice form of the receptor Fgfr2b. This upregulation of Fgfr2b in the sutures apparently causes the craniosynostotic phenotype. This hypothesis is supported by Oldridge et al. () who demonstrated ectopic expression of FGFR2b in a fibroblastic cell line derived from a patient with a mutation in the IIIc immunoglobulin-like loop of FGFR2.

It is known that in mouse calvarial explants beads impregnated with FGF4 will induce Msx1, and that FGF2 will induce Twist, a transcription factor in which loss of function mutations have been found to cause craniosynostosis (). Furthermore, FGF2 beads downregulate Fgfr2 expression by proliferating calvarial osteoblasts but upregulate Fgfr1, which is expressed by more mature calvarial osteoblasts (). Meanwhile, FGF added to the media of rat calvarial explants is known to act as a mitogen to ‘periosteal fibroblasts’ (). Iseki et al. 1999 suggest that exogenous FGF2 stimulates mesenchymal cell proliferation and differentiation in osteogenic cells (). The effects of locally applied FGFs have been further studied in relation to mouse calvarial osteoblast function. FGF2 downregulates the early osteoblastic markers alkaline phosphatase and osteonectin but will upregulate osteopontin which is a marker of more mature osteoblasts. In contrast, FGF2 also inhibits calvarial bone mineralization as visualized by alizarin red staining (). In addition, osteoblastic cell lines carrying either Apert or Crouzon mutations exhibit an inhibition of cellular differentiation and an induction of apoptosis ().

The action of FGFs in calvarial bone development are therefore far from being comprehensively understood, and it is set into this slight confusion that a recent paper by Moore et al. in Developmental Biology attempts to address some of these questions (). The authors used beads impregnated with varying concentrations of either FGF2 or neutralizing antibody to FGF2 or a combination of both, and placed them on chick calvarial explants. They were surprised to find that FGF2 beads had no effect on calvarial bone development as assessed by alizarin red staining. However, beads impregnated with neutralizing antibodies to FGF2 generated a concentration-dependent response. Instead of having a very local effect, a generalized increase in explant size together with an increase in mesenchymal proliferation was observed when one bead was used, and this had little effect on calvarial bone development. Furthermore, when three FGF2-neutralizing antibody beads were placed, the size of the explant was unchanged and calvarial bone development was severely disrupted.

Although one may know the concentration of a growth factor or antibody in which a bead is incubated, what is less certain is the concentration delivered to a tissue, the distance traveled within that tissue and over what time period the protein is effective. Both Iseki et al. () and Moore et al. () have attempted to address this by performing well-designed protein assays over a timed period. Iseki et al. () examined the spread of digoxigenin-labeled FGF2 from beads and found it to be maximal between 4 and 24 hours and reduced 48 hours after insertion. Moore et al. () assayed FGF2 by immunohistochemistry and ELISA, following the implantation of one or three beads soaked in FGF2 antibodies. They discovered that the concentration of protein released from three beads was greater than that released from a single bead and that the distance traveled was further.

The effects of FGF2 may well be differentiation stage specific (). FGF2 stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces osteocalcin production and matrix mineralization in more mature cells (). The finding that FGF2 both downregulates the late osteoblastic marker bone sialoprotein in mature osteoblasts and at the same time induces bone sialoprotein expression in less mature calvarial mesenchymal cells supports the hypothesis that FGF2 may indeed have different effects at different levels of cell maturity ().

Other Fgfs, including Fgf10, -18 and -20, have also been implicated during calvarial bone formation (,,). Fgf18 deficient mice exhibit a reduced calvarial mesenchymal proliferation rate and decreased osteoblast differentiation. This is manifest as a delay in suture closure.

Following the progress that has been made in understanding the role of Fgfs during calvarial bone development and its relation to craniosynostosis, a relatively complicated picture is emerging which involves several Fgfrs and Fgfs exerting possibly different effects on osteoblast proliferation, differentiation, function and cell death.


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