1 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot Israel
2 Institute for Animal Science, The Volcani Center, Beit Dagan, Israel
3 Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK
*Author for correspondence (e-mail: peter.lonai{at}weizmann.ac.il)
Accepted 17 May 2002
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SUMMARY |
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Key words: Transcriptional alternatives, FGF, Endochondral ossification, Craniosynostosis, Gene targeting, Mouse
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INTRODUCTION |
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Epithelial-mesenchymal interactions are fundamental to organogenesis; understanding the function of FGFR variants in the cross-talk between embryonic tissues is therefore of considerable importance. Exon-specific gene targeting revealed that the activity of Fgfr1 during late gastrulation (Deng et al., 1994; Yamaguchi et al., 1994
) is mostly due to its IIIc alternative (Partanen et al., 1998
). Recent results imply that the IIIb alternative of Fgfr1 may be active in skin development, serving as a receptor for the mesenchymal FGF10 ligand (Beer et al., 2000
). The role of Fgfr2IIIb has been studied in detail (De Moerlooze et al., 2000
; Revest et al., 2001
) (our unpublished results). Loss of Fgfr2IIIb abrogates limb outgrowth with multiple defects in branching morphogenesis. This phenotype is similar to the null mutation of Fgfr2 after rescuing its placentation defects (Arman et al., 1999
), as well as to loss of function mutation of Fgf10 (Sekine et al., 1999
).
We have analysed the functional activity of Fgfr2IIIc, the second alternative product of Fgfr2, which utilizes exon 9. This splice variant is expressed in the skeletogenic mesenchyme (Orr-Urtreger et al., 1993), and activating mutations in exon 9 of the human FGFR2 gene are associated with craniosynostosis (Wilkie, 1997
). To create a loss-of-function phenotype, we introduced a point mutation into exon 9 of Fgfr2, which caused a frame-shift and created a translational stop codon, without influencing the expression of Fgfr2IIIb. Our data show that loss of Fgfr2IIIc results in a recessive viable phenotype with craniosynostosis and retarded development of the axial and appendicular skeleton, causing dwarfism and misshapen skull. We show that the normal expression of Spp1, Cbfa1, Ihh and PTHrP (Pthlh - Mouse Genome Informatics) requires Fgfr2IIIc, consistent with the role of this receptor in maintaining the normal rate of bone formation. These results demonstrate that Fgfr2IIIc fulfills a positive role in bone development, in contrast to negative regulation by Fgfr3 (Colvin et al., 1996
; Deng et al., 1996
).
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MATERIALS AND METHODS |
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In situ hybridization
Whole-mount in situ hybridization of E16.5 fetal heads was carried out as described previously (Iseki et al., 1999). Radioactive in situ hybridization and the Fgfr2IIIb and IIIc probes were as described by Orr-Urtreger et al. (Orr-Urtreger et al., 1993
). The probe for Runx2/Cbfa1 was a gift from Dr G. Karsenty (M. D. Anderson Cancer Center, Houston, Texas), for Ihh, from A. McMahon (Dept. of MCB, Harvard University, Boston, Mass.), for PTHrP from M. Kronenberg (Mass. General hospital, Boston, Mass.) and for Spp1 from B. Hogan (Vanderbilt University, Memphis, Tenn.).
BrdU assay
Pregnant females were injected with a 10 mg/ml soulution of BrdU (Roche), at 100 µg/g body weight. Embryos were fixed in Bouins fluid and embedded in paraffin. Alternate tissue sections were stained with Mallorys trichrome for histology; the others were incubated with anti-BrdU antibody (Roche) and visualized with biotin-conjugated goat anti-mouse IgG (Vector) followed by peroxidase reaction (brown colour for BrdU-positive nuclei). These sections were counter-stained with Haematoxylin (blue nuclei). Analysis was carried out using a Kontron (Zeiss) KS400v3 automatic image analyser. A defined area including the osteoid (unmineralized matrix) of the frontal and parietal bones in the coronal sutural region, together with the preosteoblasts and osteoblasts, but excluding adjacent non-skeletogenic membrane tissues, was outlined and measured. Within equivalent areas of each of 10 wild-type and 10 mutant sections, BrdU-positive and total nuclear area was assessed by setting the detection levels for brown only or brown plus blue. Ratios were calculated for each pair of measurements and the ratios for wild-type and mutant embryos compared by the Students t-test. For each defined area, BrdU uptake was also assessed by counting the BrdU-positive cells; these counts were separately compared by the Students t-test.
Photography
A Zeiss Axioplan, a Leitz Macroscope, or a Nikon DXM1200 microscope with a CCD camera were used.
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RESULTS |
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Mice homozygous for the Fgfr2IIIc mutation (Fig. 1), are viable and fertile. They are distinguished by small size and abnormally shaped head (see later). To evaluate the specificity of the Fgfr2IIIc loss-of-function phenotype, it was important to know whether the mutation affects the expression and splicing of the alternative Fgfr2IIIb variant. In situ hybridization of sections prepared from E12.5 embryos detected Fgfr2IIIb transcripts in the surface ectoderm of the developing wild-type and Fgfr2IIIc/ limb (Fig. 2A-D). At E14.5, Fgfr2IIIb transcripts were detected in the perichondrium of prevertebrae and ribs, and in the branching epithelium of the lungs in both wild-type and Fgfr2IIIc/ embryos (Fig. 2E-H). We therefore conclude that the point mutation in exon 9 results in a phenotype specific for the IIIc splice variant of Fgfr2.
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Differentiation and growth of the skull vault
Osteopontin, which is encoded by the gene secreted phosphoprotein 1 (Spp1), is one of the major non-collageneous bone matrix proteins, produced by osteoblasts and osteoclasts. It is copiously expressed by mineralized bone and is involved in bone remodelling. Hence Spp1 expression is a good indicator of osteogenesis and can be used to detect the developing bone domains in the skull vault by means of whole-mount in situ hybridisation (Iseki et al., 1997). At E16.5, Spp1 expression revealed a delay in expansion of the frontal bone domains towards the midline to form the metopic suture; the parietal and interparietal domains were normal, but the nasal bone domains were undetectable (Fig. 7A,B). In alizarin-stained specimens at P3 the pattern of skull vault bones was not detectably different in mutant and wild-type pups (not shown). By P14, the metopic, sagittal and lambdoid sutures of both mutant and wild-type skulls were unfused, but the mutant coronal suture showed partial (medial) or complete bony fusion in some specimens (Fig. 7C-F). These observations suggest that there is a catch-up following the late onset of ossification seen in E14.5 specimens, and that this more rapid rate of ossification in the mutants continues as premature loss of the coronal suture in at least some pups by P14. Except for the coronal suture, morphogenesis of the skull vault was less affected than that of the skull base.
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The effects of loss of Fgfr2IIIc function on endochondral differentiation
During endochondral bone formation and growth, proliferating chondrocytes in the diaphysis of the cartilaginous model at early stages, and in the growth plates throughout the period of bone growth, undergo hypertrophy and finally apoptosis. They produce a framework of cartilage matrix, which forms a template for the deposition of bone matrix proteins by invading osteoblasts. The first ossification is in the perichondrium, which is transformed into the periosteal collar of bone around the diaphysis, before the primary (diaphyseal) ossification center has formed. To gain insight into the effects of loss of Fgfr2IIIc function on endochondral bone differentiation, we investigated gene expression in the skull base at E18.5 and P7, and in the tibial growth plate at P7 and P14. Transcript levels of the osteogenic differentiation marker Spp1, which is expressed in the differentiating osteoblasts, were reduced in the regions of ossification of both the skull base (Fig. 9A-D) and the tibia at P14 (Fig. 9E,F), confirming that Fgfr2IIIc affects endochondral bone formation in both the skull base and the long bones.
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DISCUSSION |
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Fgfr2IIIc/ mutants displayed normal limb development, the reduced length of the limb bones being proportional to the reduced size of the whole skeleton. This phenotype is distinct from that caused by loss of the IIIb alternative. We prepared a similar frame shift mutation in exon 8 of Fgfr2IIIb (V. P. E. and P. L., unpublished), which resulted in the limbless phenotype with defective branching morphogenesis as reported for Fgfr2IIIb/ embryos by Revest et al. (Revest et al., 2001). Fgfr2IIIb is localized in the apical ectodermal ridge (AER) of the limb bud (Orr-Urtreger et al., 1993
); this receptor isoform interacts with FGF10 ligand in the progress zone mesenchyme (Ohuchi et al., 1997
; Xu et al., 1998
), whereas Fgfr2IIIc transcripts are detectable in the limb bud mesenchyme. The normal limb development observed in the Fgfr2IIIc loss of function mutation suggests that this receptor may not be the partner of AER-derived FGFs. The IIIc variant of FGFR1 is a more probable receptor for these growth factors, since mutation of this receptor isoform results in a hypomorphic limb phenotype (Partanen et al., 1998
).
A number of mutations of the IIIc alternative of FGFR2 and FGFR1 are associated with different types of human craniosynostosis syndromes. It is generally accepted that most FGFR mutation-derived dominant human craniosynostoses are due to gain of function resulting from ligand-independent signaling (for review, see Wilkie, 1997). Craniosynostosis in our mutation was distinguished from the human gain of function phenotypes by its predominant manifestation in the endochondral skull base, and by its recessive inheritance.
The occurrence of synostosis of the coronal suture in both the murine Fgfr2IIIc loss-of-function mutation demonstrated here and in human gain-of-function mutation in the same splice variant, such as the Crouzon syndrome, appears at first sight to be paradoxical, but is a logical outcome of the mechanism of activity of this receptor. Functional studies in the mouse revealed that Fgfr1 and Fgfr2 play reciprocal roles in maintaining the proliferation-differentiation balance in osteoprogenitor cells of the coronal suture, and are expressed respectively in differentiating and proliferating cells (Iseki et al., 1999). The proliferation-differentiation balance is maintained by differential effects of high and low levels of FGF-FGFR signaling. Adding exogenous FGF2 ligand to the developing coronal suture results in up-regulation of Fgfr1 and increased osteogenic differentiation, with concomitant down-regulation of Fgfr2 and decreased osteogenic cell proliferation. The progressive loss of osteogenic proliferating cells in the coronal suture in the absence of Fgfr2IIIc, as reported here, is consistent with this model.
Fgfr2IIIc also plays a role in endochondral ossification. The onset of mineralization was retarded in our mutants, and the growth of the skull base and axial and appendicular skeletons was reduced. This outcome was associated with decreased areas of both proliferating chondrocytes and ossification zones in these endochondral bones, leading to premature loss of skull base sutures and smaller than normal long bones and vertebrae. Consistent with these observations, we found a significant decrease in the accumulation of transcripts characteristic of chondrocytes and osteoblasts, suggesting that both require Fgfr2IIIc function. This transcriptional alternative is expressed in the mesenchymal condensations that form the cartilage models of endochondral bones. Later, its transcription shifts to the perichondrium as it transforms into the periosteal bone collar at the onset of osteogenesis, and then in the developing trabecular bone of the ossification zone. It is not clear how Fgfr2IIIc in the ossification zone influences chondrocyte proliferation in the growth plate. It is possible that this effect is the result of events occurring during its early mesenchymal expression, when Fgfr2IIIc could affect common precursors of both lineages and thus influence the size of cartilage models, the onset of differentiation and the long term process of endochondral bone growth. Alternatively, the primary target of Fgfr2IIIc may be the osteocyte lineage through activation of Runx2/Cbfa1, which has a major role in osteogenesis (Ducy et al., 1997) but also influences chondrogenesis (Kim et al., 1999
; Takeda et al., 2001
).
Recent publications by Liu et al. (Liu et al., 2002) and Ohbayashi et al. (Ohbayashi et al., 2002
) investigated the loss-of-function phenotype of Fgf18. Both papers report that the phenotype resembles the Fgfr3 loss-of-function phenotype resulting in extended long bone growth, but also delayed ossification with decreased expression of osteogenic markers. They suggest that FGF18 signaling co-ordinates chondrocyte and osteoblast differentiation. The evidence we present here suggests that the FGFR2IIIc receptor, which together with FGFR3IIIc binds FGF18, may contribute to this co-ordinated regulation of osteogenesis.
The expression pattern of Fgfr2IIIc in developing endochondral bones is different from those of Fgfr1 and Fgfr3. Fgfr1 is expressed in the perichondrium during early bone formation, but is later transcribed by proliferating and hypertrophic chondrocytes of the growth plate (Orr-Urtreger et al., 1991; Peters et al., 1992
; Delezoide et al., 1998
). Fgfr3 transcription localizes to the resting and proliferating chondrocyte layers (Deng et al., 1996
) and its loss results in long bone overgrowth with massive extension of the proliferating chondrocyte layer (Peters et al., 1993
; Deng et al., 1996
; Colvin et al., 1996
). Gain-of-function mutations of Fgfr3 create the opposite phenotype. They cause achondroplasia in man and their molecular effects include repression of Ihh and PTHrP signaling (Naski et al., 1998
) and activation of cell cycle inhibitors (Li et al., 1999
; Sahni et al., 1999
). This is in contrast to the dwarfism and decrease of Spp1, Ihh, PTHrP and Cbfa1 expression in our Fgfr2IIIc loss-of function mutation. This contrast is emphasised in a gain-of-function mutation of Fgfr2IIIc with a C342Y amino acid replacement in exon 9, in which Cbfa1/Runx2 and Spp1 transcription is activated (V. P. E., G. M. M.-K. and P. L., unpublished).
Comparison of the role of FGFRs in bone development suggests that the rate of proliferation and differentiation of skeletogenic precursors involves cooperation between different FGFRs. In the coronal suture, synostosis can result from gain-of-function mutations of FGFR1, FGFR2 and FGFR3 (Bellus et al., 1996). Although the splice variants of Fgfr1 and Fgfr3 are yet to be studied, these genes show specific expression patterns and function in the coronal suture (Iseki et al., 1999
; Johnson et al., 2000
), in endochondral osteogenesis (Peters et al., 1993
; Orr-Urtreger et al., 1993
; Delezoide et al., 1998
) and in developing dental tissue (Kettunen et al., 1998
).
The present results extend these data, suggesting a balanced cooperation between the negative regulation due to Fgfr3 and the positive control exerted by Fgfr2IIIc. The data of Liu et al. (Liu et al., 2002) and Ohbayashi et al. (Ohbayashi et al., 2002
), discussed above, suggest that this co-ordination of chondrocyte and osteoblast differentiation is mediated through FGF18. All three of the alternatively spliced Fgfr genes are active during early osteogenesis and influence the expression of multiple bone development genes. Hence, gene expression and lineage decisions required for osteogenesis may depend on their balanced cooperation. The mode of this control, whether conveyed by transcriptional regulation and/or by indirect means involving the bone matrix, remains to be elucidated.
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ACKNOWLEDGMENTS |
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