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Human Molecular Genetics Advance Access originally published online on December 17, 2003
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Human Molecular Genetics, 2004, Vol. 13, No. 3 271-284
DOI: 10.1093/hmg/ddh034

Defective bone mineralization and osteopenia in young adult FGFR3–/– mice

Gladys Valverde-Franco1,2,3, Hanlong Liu1,2,3, David Davidson1,2,3, Sen Chai1,2, Hector Valderrama-Carvajal1,4, David Goltzman1,2,5, David M. Ornitz6 and Janet E. Henderson1,2,3,*

1Department of Medicine and 2Centre for Bone and Periodontal Research, McGill University, Montreal, Quebec, Canada, 3Division of Endocrinology, 4Division of Molecular Endocrinology and 5Calcium Research Laboratory, McGill University Health Centre, Royal Victoria Hospital, Montreal, Quebec, Canada and 6Washington University School of Medicine, St Louis, MO, USA

Received August 19, 2003; Accepted December 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Histomorphometry
 RT-PCR analysis of molecular...
 REFERENCES
 
Mutations that cause constitutive activation of fibroblast growth factor receptor 3 (FGFR3) result in skeletal disorders that are characterized by short-limbed dwarfism and premature closure of cranial sutures. In previous work, it was shown that congenital deficiency of FGFR3 led to skeletal overgrowth. Using a combination of imaging, classic histology and molecular cell biology we now show that young adult FGFR3–/– mice are osteopenic due to reduced cortical bone thickness and defective trabecular bone mineralization. The reduction in mineralized bone and lack of trabecular connectivity observed by micro-computed tomography were confirmed in histological and histomorphometric analyses, which revealed a significant decrease in calcein labelling of mineralizing surfaces and a significant increase in osteoid in the long bones of 4-month-old FGFR3–/– mice. These alterations were associated with increased staining for recognized markers of differentiated osteoblasts and increased numbers of tartrate-resistant acid phsophatase postitive osteoclasts. Primary cultures of adherent bone marrow-derived cells from FGFR3–/– mice expressed markers of differentiated osteoblasts but developed fewer mineralized nodules than FGFR3+/+ cultures of the same age. Our observations reveal a role for FGFR3 in post-natal bone growth and remodelling, which identifies it as a potential therapeutic target for osteopenic disorders and those associated with defective bone mineralization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Histomorphometry
 RT-PCR analysis of molecular...
 REFERENCES
 
FGFR3 is one of four structurally and functionally related receptors, which transduces signals from many of the 22 identified fibroblast growth factor (FGF) polypeptides (1,2). Binding to the high-affinity FGFR is facilitated by heparan sulfate proteoglycan (HSPG) and results in FGFR dimerization and auto-phosphorylation. Receptor activation initiates signalling through transduction pathways linked to ras-raf-MEKK (3), phospholipase-C{gamma} (PLC {gamma}) (4) and STAT-1 (5). FGFR signalling regulates cell proliferation, differentiation and survival in a time- and tissue-specific manner. Restricted expression (6) and alternative splicing of the receptor (7), restricted expression of heparan sulfate proteoglycan (8 and other reviews, 9), heterodimerization with other family members (10) and processing to generate soluble receptors (11) have all been proposed to contribute to the diverse biology attributed to FGFR signalling.

Most bones of the skeleton develop by a process of endochondral ossification, during which a cartilage template is mineralized and replaced by bone. In contrast, bones of the cranial vault develop by intramembranous ossification, which does not involve a cartilage intermediate. In bones formed by endochondral ossification FGFR2 has been identified in the perichondrium, the periosteum and the metaphysis, whereas FGFR3 is expressed predominantly by resting and proliferating chondrocytes. FGFR1 is expressed by hypertrophic chondrocytes (12–14), in the periosteum and in metaphyseal osteoblasts (15). In the developing skull FGFR1 shares an overlapping expression domain with FGFR3 in the periosteum and with FGFR2 in the osteogenic fronts of developing sutures, where it has been implicated in craniosynostosis syndromes (16,17).

The role of FGF signalling in the developing skeleton was confirmed when several autosomal dominant human osteochondrodysplastic disorders were mapped to point mutations in FGFR3 (17–19). More than 50 such mutations have been identified to date. Those associated with FGFR3, such as achondroplasia (ACH), hypochondroplasia (HCH) and the thanatophoric dysplasias (TDs), preferentially affect long bone development. Affected individuals exhibit defects in cortical and trabecular bone, which are often associated with altered vascularization (20). Craniosynostosis syndromes, on the other hand, are associated primarily with defective FGFR2 signalling.

Targeted mutation of the mouse gene encoding FGFR3 has further clarified its role in endochondral bone development (13,21,22). Mice homozygous for null mutations in FGFR3 survived but developed skeletal overgrowth, which was attributed in part to increased proliferation and accumulation of chondrocytes in the growth plates of developing bones. In contrast, mice carrying different activating mutations in FGFR3 were unfailingly dwarfed, and had reduced numbers of proliferating and hypertrophic chondrocytes in their growth plates (23–26). Taken together, these observations implicate FGFR3 in the negative regulation of endochondral bone growth.

Of the 22 potential ligands for FGF receptors, FGF2 and FGF18 have been identified as important mediators of their biological activity in the skeleton in vivo. FGF2 is expressed in osteoblasts in bone (27) and in growth plate chondrocytes (28). At birth, the skeletons of mice homozygous for targeted inactivation of FGF2 appear normal suggesting that other ligands signal through FGFR3 during development (29). However, by 5 months of age the FGF2-deficient mice had a significant reduction in trabecular bone mass that was due to reduced formation, rather than to increased resorption. FGF18 is expressed in the perichondrium and periosteum of developing long bones, in developing joints and in osteogenic cells of the calvarium (30,31). In contrast to the lack of developmental defects in the FGF2 null mice, those lacking FGF18 died shortly after birth with skeletal abnormalities similar to those seen in mice lacking FGFR3. Additional defects in FGF18 null mice, which were not apparent in FGFR3 null mice, included delayed closure of cranial sutures and delayed ossification of metatarsal bones. These data suggest that FGF18 signals to FGFR3 in developing chondrocytes but also to other FGFRs throughout developing bone.

Previous work has demonstrated that FGFR3 is an important mediator of chondrocyte function during endochondral bone development and of osteoblast function during intramembranous bone development but its role in post natal bone growth remains poorly defined. In the current work we showed that young adult mice lacking functional FGFR3 were osteopenic and developed severe osteomalacia. These defects were not due to alterations in PTH or vitamin D metabolism, or to a deficiency of mature osteoblasts, but were associated with altered expression of other FGF receptors and osteoblast differentiation markers. These results demonstrate an important role for FGFR3 signaling in osteoblast biology and identify it as a potential biomarker for adult skeletal disorders.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 Histomorphometry
 RT-PCR analysis of molecular...
 REFERENCES
 
Phenotypic analysis of 4-month-old mice
In previous studies FGFR3–/– mice maintained on a C57Bl6 background failed to thrive. Fifty percent mortality occurred by the age of 1 month, which may have been due in part to the development of severe kypho-scoliosis and difficulty with breathing (13,21). To enable investigation of post-natal bone growth in FGFR3–/– mice in the absence of these confounding factors, C57Bl6 mice heterozygous for targeted disruption of FGFR3 were out-crossed to a C3H background. After 10 generations, 90% of the FGFR3 null mice survived past 6 months of age. Four-month-old FGFR3–/– mice demonstrated minimal kyphosis and similar body size to age matched controls (Fig. 1A), although the vertebrae and limbs, visualized by whole body X-ray, were significantly longer in the FGFR3–/– mice (Fig. 1B). Morphometric data, shown in Table 1, indicate that there was no significant reduction in weight of the mutant mice at 2 months, and only a 15% reduction at 4 months, compared with FGFR3+/+ littermates. As shown previously (21), there were significant increases in all bone lengths at both ages, which confirmed that the skeletal phenotype was not altered by the change from C57Bl6 to C3H background. Clear radiological evidence of bone and joint abnormalities was evident as early as 2 months and worsened by 4 months, in FGFR3–/– mice (Fig. 2). In addition to mis-shapen bones, abnormal articulation and generalized osteopenia, the mutant mice exhibited profound thinning of cortical bone (Fig. 2B, arrowhead). To confirm the X-ray findings the bone mineral density (BMD) of FGFR3–/– mice and FGFR3+/+ littermates was also assessed at 2 months and 4 months of age. Table 2 shows a small reduction in BMD in the mutant mice at 2 months, which apparently resolved at 4 months. However, owing to the difficulties in positioning the bent limbs of FGFR3–/– mice, it was decided to scan isolated femurs, which then showed a highly significant reduction in BMD. This result demonstrates the need for caution when scanning intact mice with significantly different bodily architecture. Having demonstrated a reduction in bone in the limbs of mature FGFR3–/– mice using two independent measurements we next examined the architecture and biomechanical strength of the bones.



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  		Figure 1. Morphologic and radiologic assessment of 4-month-old FGFR3+/+ and FGFR3–/– littermates. FGFR3–/– mice out-crossed for 10 generations onto a C3H background looked similar in size and appearance to their wild-type littermates, except for mild kyphosis and a kinky tail (A). FGFR3–/– mice demonstrated elongation and curvature of the axial and appendicular skeleton (B, brackets) similar to that seen previously in mice on a C57Bl6 background. The phenotype and whole body radiographs are representative of 10–15 animals of each genotype.

 

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  		Table 1. Morphometric analysis of skeletal elements of FGFR3 +/+ and FGFR3–/– mice at 2 and 4 months of age
 


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  		Figure 2. Faxitron X-ray analysis of FGFR3+/+ and FGFR3–/– littermates at 2 and 4 months. High resolution X-ray analysis of the femoral-tibial junction of representative FGFR3+/+ (left) and FGFR3–/– (right) mice at 2 months (A and B) and 4 months (C and D) of age revealed significant deformation of the knee joint, generalized osteopenia and thinning of cortical bone (arrowheads) in the mutant mice. Radiographs are representative of 10–15 mice of each genotype.

 

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  		Table 2. Bone mineral density of FGFR3+/+ and FGFR3–/– mice at 2 and 4 months of age measured by Piximus® densitometer
 
Micro-architecture and biomechanical strength of femur from 4-month-old mice
Micro-architectural changes in the femora and vertebra of 4-month-old FGFR3–/– mice and FGFR3+/+ littermates was assessed by micro CT and the biomechanical strength measured by three-point bending. Reconstruction (Fig. 3A and B) and three-dimensional rendering (Fig. 3C and D) of scans of the distal femoral metaphysis confirmed the reduction in cortical thickness seen by X-ray (Fig. 2B, arrowhead). Furthermore, there was a decrease in trabecular thickness and connectivity in FGFR3–/– mice (Fig. 3D). These changes in bone architecture were confirmed by quantitative analysis of the femoral metaphysis and the fourth lumbar vertebra. The femoral metaphysis revealed a significant reduction in the percentage of thick trabeculae in FGFR3–/– mice (Fig. 3E, dotted line), which was reflected in an overall decrease in mean trabecular thickness, shown in Table 3. The percentage bone (mineralized tissue) was decreased by 14% in the femoral metaphysis and by 47% in the fourth lumbar vertebra and the visible loss of trabecular connectivity was confirmed by a significant increase in the structure model index (SMI) in FGFR3–/– femora and vertebra compared with littermate controls.



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  		Figure 3. Micro-CT analysis of the distal femur of 4-month-old FGFR3+/+ and FGFR3–/– littermates. Representative two-dimensional (A and B) and three-dimensional (C and D) reconstructions, obtained with a 0.9° rotation between frames on a Skyscan® 1072 instrument, are shown for FGFR3+/+ (left) and FGFR3–/– (right) mice. The upper panels are representative of the area just below the growth plate while the lower panels show the three-dimensional reconstruction from 200 adjacent images extending into the metaphyseal bone. FGFR3–/– mice exhibited profound thinning of cortical bone (B, arrowhead), a reduction in platelike structures and a lack of trabecular connectivity (D). Quantitation of trabecular thickness distribution (E) further exemplified an increase in the number of thin trabeculae in the mutant femora (hatched line) compared with the FGFR3+/+ littermate control (solid line). Results are expressed as the mean±SD of eight independent analyses per group. Significantly different from control; *P<0.05, **P<0.01.

 

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  		Table 3. Quantitative micro CT analysis of the femoral metaphysis of 4-month-old FGFR3+/+ and FGFR3–/– mice
 
The biomechanical strength and resilience of freshly dissected femora from 4-month-old FGFR3+/+ and FGFR3–/– mice was evaluated at room temperature by three-point bending using a Mach-1® tester. The decrease in cortical width and compromised architecture of FGFR3–/– femora led to a reduction in the maximum load to failure (FGFR3+/+ 33.83±6.58 versus FGFR3–/– 23.74±4.37 N, P<0.001), an increase in their elasticity, or displacement to maximum load (FGFR3+/+ 332.56±34.28 versus FGFR3–/– 458.25±96.30 µm, P<0.001) and a reduction in their rigidity (161.02±22.74 versus FGFR3–/– 104.32±26.06 N/mm, P<0.001). The changes in mass and architecture were therefore accompanied by a reduction in the mechanical strength of FGFR3–/– femora.

Using quantitative, non-invasive imaging techniques and bone densitometry it was thus shown that 4-month-old male FGFR3–/– mice had a reduced bone mass with lower mineral content and compromised architecture compared with their FGFR3+/+ littermates. These changes in mass and architecture were accompanied by a reduction in biomechanical strength.

Histologic analysis of undecalcified and decalcified tibiae from 4-month-old mice
To examine the molecular and cellular basis for the osteopenic phenotype of FGFR3–/– mice we performed histological analyses on undecalcified and decalcified tibia. Von Kossa stained sections revealed a significant decrease in the width of cortical bone and an increased width of fibrous tissue adjacent to the periosteum in FGFR3–/– mice compared with wild-type littermates (Fig. 4A and B, asterisk). In FGFR3+/+ mice the endosteal surfaces and trabecular bone (Fig. 4C and G arrow) were lined with thin osteoid seams and bordered by flattened osteoblasts, whereas those of FGFR3–/– mice were lined with thick osteoid seams bordered by large, cuboidal osteoblasts (Fig. 4D and H, arrow). Histomorphometric analyses of undecalcified sections from 2- and 4-month-old mice (Table 4) revealed a similar modest decrease in the percentage bone in the FGFR3–/– tibia, as seen by quantitative micro CT. However there was a 7- to 10-fold increase in osteoid volume, a 2.6- to 3.8-fold increase in osteoblasts and a 2.6- to 2.9-fold increase in osteoclasts.



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  		Figure 4. Histochemical analysis of undecalcified bone. The proximal tibia of 4-month-old FGFR3+/+ (left) and FGFR3–/– (right) mice were left un-decalcified and embedded in a mixture of MMA and GMA. Two micron sections were stained with von Kossa to show mineral deposits and counterstained with toluidine blue to show osteoid and fibrous tissue. FGFR3–/– mice consistently exhibited thinner cortices bordered by thick fibrous tissue at the periosteal surface (B, asterisk) compared with FGFR3+/+ control (A, asterisk). The endosteal and trabecular surfaces of FGFR3–/– bones were lined by thick osteoid and large osteoblasts (D and H arrow) compared with control (C and G arrow). Magnification in (A), (B), (E) and (F) x20, in (C), (D), (G) and (H) x40. Micrographs are representative of those from five different mice of each genotype.

 

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  		Table 4. Quantitative histomorphometric analysis of the tibial metaphysis of 2- and 4-month-old FGFR3+/+ and FGFR3–/– mice
 
The similar histological phenotypes in 2- and 4-month-old FGFR3–/– mice suggested there was a primary defect in osteoblast function in the mutant mice. We therefore examined the FGFR3+/+ and FGFR3–/– bones for recognized molecular markers of cellular activity. In situ staining revealed increased numbers of ALP positive osteoblasts (Fig. 5A and B blue, arrowheads) and TRAP positive osteoclasts (Fig. 5A and B red, arrows) lining trabecular bone surfaces in FGFR3–/– mice compared with littermate FGFR3+/+ controls. Concommitant with the increased osteoblast numbers, osteocalcin (Fig. 5C and D, arrows) and matrix metalloproteinase (MMP-13; Fig. 5E and F arrows) immunoreactivity were both up-regulated in the FGFR3–/– tibia. Despite this increase in mature osteoblasts, calcein labelling of the mineralization fronts in FGFR3–/– mice was reduced compared with their littermate controls (Fig 5G and H, yellow).



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  		Figure 5. Staining for maturation markers and matrix mineralization in 4-month-old mice. Sections of plastic embedded tibiae from FGFR3+/+ (left) and FGFR3–/– (right) were stained sequentially for ALP (osteoblasts) and TRAP (osteoclasts) and sections from the decalcified, paraffin-embedded contra-lateral tibiae stained with antibodies raised against markers of osteoblast maturation. A significant increase in ALP (A and B, blue arrowheads) and TRAP (A and B, red arrows) enzyme activity was seen in FGFR3–/– mice (right) compared with FGFR3+/+ (left) mice. Immunoreactivity for OCN and MMP-13, which are markers of mature osteoblasts, were also increased in the FGFR3–/– bone (D and F, arrows) compared with FGFR3+/+ control mice (C and E, arrows). Labelling for calcein, which was administered IP prior to sacrifice to identify mineralization fronts, was significantly reduced in the FGFR3–/– tibia (H) compared with that of the FGFR3+/+ control (G). Magnification x20. Micrographs are representative of those from five different mice of each genotype.

 
The combination of numerous, cuboidal, ALP positive osteoblasts on thick osteoid, decreased tetracycline labelling and up-regulation of differentiation markers such as osteocalcin and MMP-13 in the FGFR3–/– mice suggested that the osteomalacic phenotype was due primarily to a failure to mineralize the matrix rather than to defective osteoblast differentiation.

Serum biochemistry
To confirm that the bone defects were not due to systemic changes in mineral ion homeostasis we examined circulating levels of hormones and markers of mineral ion metabolism. Table 5 shows no significant differences in serum or urine levels of Ca and PO4, or serum levels of PTH, 25(OH)D or 1,25(OH)2D between the control and mutant mice. In contrast there was a significant elevation in circulating ALP levels in the FGFR3–/– mice, which most probably reflected the increase in osteoblastic activity in these mice. These observations further supported the hypothesis that FGFR3 deficiency in young adult mice resulted in a localized defect in bone mineralization.


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  		Table 5. Biochemical analysis of serum and urine from 4-month-old FGFR3+/+ and FGFR3–/– mice.
 
Bone marrow stromal cell cultures
To further explore the molecular basis for the defective mineralization in FGFR3–/– bones we established primary cultures of bone marrow stromal cells to examine cellular and molecular markers of proliferation and differentiation. Using an MTT cell viability assay it was shown that sub-confluent FGFR3–/– stromal cell cultures grew more rapidly under basal conditions and were more responsive to FGF 2 and FGF18 stimulation (Fig. 6A). Cultures of FGFR3–/– cells maintained for 3, 6 and 18 days in differentiation medium stained more intensely for ALP at 3 and 6 days but contained fewer von Kossa positive mineralized nodules at 18 days (Fig. 6B).



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  		Figure 6. Growth and differentiation of cultured stromal cells from FGFR3+/+ and FGFR3–/– mice. Adherent bone marrow cells flushed from the tibia and femora were maintained for up to 18 days in culture medium containing 50 µg/ml ascorbic acid, 10 mM ß-glycerophosphate and 10–8 M dexamethasone. Using an MTT assay (A) it was shown that FGFR3–/– cells (hatched bars) had a higher basal growth rate and were more responsive to FGF stimulation than FGFR3+/+ cells (white bars). Quantitative analysis of the ALP activity and mineral deposition, shown on the left in (B), revealed increased staining for ALP at all time points in FGFR3–/– cultures but fewer von Kossa positive mineralized nodules at D18 compared with control. Results are representative of three independent experiments and are expressed as the mean±SD of four replicates per group. Significant difference from control, **P<0.01.

 
Semi-quantitative RT–PCR analysis of RNA harvested from bone marrow stromal cells after 6 and 18 days of culture (Fig. 7) showed that those established from FGFR3+/+ mice expressed FGFR3 at a low level, but not FGFR1 and FGFR2, at day 6. In contrast, all three receptors were strongly expressed in differentiated cells at day 18. In cultures established from FGFR3–/– mice, FGFR1 and FGFR2 were expressed prematurely at 6 days, and at similar or higher levels compared with FGFR3+/+ control at 18 days. A similar pattern was seen for IHH mRNA, which was increased in FGFR3–/– cultures compared with control at 6 days, despite similar expression of PTHrP and PTH1R. Expression levels of type I collagen, osteopontin and osteocalcin mRNA in FGFR3–/– cultures were not significantly different from those in FGFR3+/+ cultures, whereas MGP was prematurely upregulated. These results from in vitro experiments therefore confirmed the hypothesis that FGFR3 is expressed by cells of the osteoblast lineage in metaphyseal bone and suggest that its absence may promote premature differentiation but inhibit terminal differentiation and matrix mineralization.



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  		Figure 7. Gene expression profile of cultured stromal cells at D6 and D18 of culture. Total RNA was isolated at D6 and D18 from three independent cultures of cells established from FGFR3+/+ or FGFR3–/– bone marrow as described in Materials and Methods. RNA was subjected to reverse transcription and conventional PCR after optimal conditions were established for each set of primers. Data from scanning densitometric analyses is expressed as the ratio of the gene of interest to GAPDH and represents the mean±SD of triplicate determinations for FGFR3+/+ (white columns) and FGFR3–/– (black columns). A significant increase in FGFR1, FGFR2 and IHH expression was seen in cultures of FGFR3–/– cells at D6, whereas expression of PTHrP and its receptor, PTH1R were no different from FGFR3+/+ cells. MGP was also up-regulated in FGFR3–/– cells at D6 whereas type I collagen, OPN and OCN expression levels were no different from control. *P<0.05, **P<0.01 compared with wild-type control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 Histomorphometry
 RT-PCR analysis of molecular...
 REFERENCES
 
Although FGFR3 has been identified in cells of the osteogenic lineage, its role in regulating osteoblast function in post natal bone growth and remodelling in the appendicular skeleton has not yet been defined. In the current studies, we showed that the two most conspicuous features of the skeletal phenotype of 4-month-old FGFR3–/– mice were decreased cortical bone thickness and osteomalacia, both of which contributed to osteopenia and increased susceptibility to femoral fracture. This work identifies FGFR3 as an important regulator of bone development and remodelling and as a potential molecular target for osteopenic disorders and those associated with defective skeletal mineralization.

Regulation of cortical bone growth by FGF signalling
The majority of the adult mammalian skeleton is comprised of cortical bone, which is concentrated in the diaphyses of long bones. It is formed when mesenchymal cells in the periosteum differentiate directly into bone forming osteoblasts (32,33). Lifelong renewal of cortical bone in humans is believed to result by periosteal deposition, remodelling within the Haversion system of the bone matrix and resorption at the endosteal surface (34). Although mice do not have clearly defined Haversion systems (35), it is evident that osteocytes embedded in bone communicate with one another and with the bone surface to transduce signals that result in bone metabolism (36–38).

Using radiographic, micro CT and histological analyses, we demonstrated a 50% reduction in cortical bone width, accompanied by a thickened fibrous periosteum, in young adult male FGFR3–/– mice. The decrease resulted in a greater displacement on loading, hence the curvature of femora and tibiae, but also an increase in fragility, as evidenced by the decrease in maximum load to failure in the three-point bending test. Some mechanistic insight into signalling by FGFs in intramembranous bone came from recent work characterizing the skeletal phenotypes of FGF18–/– (30,31) mice and in those with conditional inactivation of FGFR2 in the skeleton (15). In developing long bones, FGF18 and FGFR2 are both highly expressed in perichondrial and periosteal tissue. FGF18 is proposed to act as an endogenous ligand for FGFR3 in growth plate chondrocytes, and for FGFR2 in osteoblasts and their precursors in the perichondrium and periosteum. FGF18–/– mice had a growth plate morphology similar to that seen in FGFR3–/– mice and exhibited delayed closure of the cranial sutures and impaired terminal differentiation of calvarial osteoblasts (31). Our current observations of reduced cortical thickness in the long bones of juvenile and young adult FGFR3–/– mice support the hypothesis that FGF18 is also a ligand for FGFR3 in osteogenic cells in the periosteum and that FGF18/FGFR3 interactions are required for cortical bone growth. Mice with targeted inactivation of FGFR2 in chondrocytes and osteoblasts (FGFR2cko) also exhibited a reduction in cortical bone width (15). However, the surrounding fibrous cell layer was decreased in the FGFR2cko mice, whereas that in FGFR3–/– mice was increased. There was also a noticeable reduction in the number and activity of osteoblasts in the juvenile FGFR2cko mice compared with the significant increase in osteoblast numbers and activity in the adult FGFR3–/– bones. Taken together these data implicate FGFR2 and FGFR3 in the development and maintenance of cortical bone and suggest that their signaling pathways may have specific and redundant functions in this respect.

An alternative explanation for cortical osteopenia in the FGFR3–/– mice arises from a recent report that showed signaling by FGF2, and by activated FGFR2, blocked ALP accumulation and nodule mineralization in primary cultures of calvarial osteoblasts (39). Endogenous FGF18 or FGF2 could have interacted with FGFR1 or FGFR2, both of which appeared to be up-regulated in FGFR3–/– mice, to inhibit osteogenesis. However, we demonstrated an increase in osteoblast numbers and activity in FGFR3–/– mice in vivo and in stromal cell cultures derived from their bones. These observations support those of others who showed that signaling through FGFR2 stimulates proliferation of early osteoprogenitor cells and activation of FGFR1 promotes their early differentiation (14,40–44). Furthermore, FGF2 has been shown to stimulate bone formation in vivo (45) and mice homozygous for targeted disruption of FGF2 developed osteopenia as a consequence of a reduction in the number and activity of osteogenic cells (29), similar to the situation seen in FGFR2 conditional knock-out mice (15).

The reduction in cortical bone in FGFR3–/– mice could also have been due to increased resorption resulting from an increase in TRAP positive osteoclasts. Osteoclasts express FGFR1 (46) and FGF2 and FGF18 have been shown to stimulate osteoclasts in vitro by direct and indirect mechanisms (46,47). Furthermore, FGF2-null mice demonstrated impaired osteoclastic activity in response to catabolic agents in vivo and in vitro (48). In FGFR3–/– mice, FGFR1 expression could have been up-regulated in osteoclast precursors, as it was in bone marrow derived stromal cells, thus contributing to increased osteoclast recruitment and activity. Alternatively, the significant increase in osteogenic cells could have promoted signaling through the RANK/RANKL axis to increase the number of osteoclasts, or changed the expression ratio of OPG/RANKL to favor osteoclastogenesis (49). Regardless of the mechanism, it is unlikely that this would have contributed significantly to cortical thinning in FGFR3–/– mice because of the presence of thick osteoid, which is believed to be resistant to osteoclastic resporption, on the majority of endosteal surfaces in the mutant bones.

FGFR3 and bone mineralization
In humans, osteomalacia is characterized by a significant accumulation of un-mineralized osteoid, a reduction in mineralized bone volume, and is often seen in the presence of plump, cuboidal osteoblasts (50). It is associated with a wide variety of disorders of phosphate metabolism that include X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), tumor-induced osteomalacia (TIO), McCune Albright syndrome and chronic renal insufficiency, as well as hypophosphatasia, aluminium intoxication and malnutrition. One hypothesis that is currently under intense investigation proposes that osteomalacia in hypophosphatemic disorders is secondary to a renal phosphate leak caused by high circulating levels of a phosphaturic agent, recently identified as FGF-23 (51–55).

Radiologic, histologic and histomorphometric analysis of the bones of juvenile and adult FGFR3–/– mice revealed decreased bone mineral density, an increase in unmineralized tissue and increased numbers of osteoblasts and osteoclasts in metaphyseal bone. It could be argued that an increase in osteoclast number and activity released factors such as IGF-1, TGFß or FGF from the bone matrix, which in turn stimulated osteogenic cell proliferation, but perhaps inhibited their capacity to mineralize bone (47,56). This increase in bioavailability, combined with altered expression levels of FGF receptors in FGFR3–/– osteoblasts and osteoclasts, could have impacted negatively on the complex interactions between their signaling pathways in these cells (22,57,58). Similarly, the relative abundance or bioactivity of a number of cytokines that impact on the anabolic and catabolic activity of skeletal cells could have been altered in the bone marrow microenvironment of FGFR3–/– mice (59).

Osteomalacia in the presence of active osteoblasts was also demonstrated in mice with XLH (60) and in those with targeted ablation of the 25 hyroxyvitamin D 1{alpha} hydroxylase enzyme gene (61,62). However, those animals were dwarfed and had abnormal circulating levels of PTH, vitamin D, calcium and phosphate. In contrast, FGFR3–/– mice demonstrated elongation of the skeleton and normal mineral ion homeostasis. Despite systemic phosphate homeostasis, it is possible that phosphate metabolism was disrupted at the local level in FGFR3–/– osteoblasts resulting in impaired mineralization. Expression and or activity of the Glvr-1 phosphate transporter is regulated by FGF and has been implicated in the early commitment of chondrogenic (63) and osteogenic cells (64) to mineralization competence. FGFR3 deficiency might also alter expression and or activity of the Phex enzyme or its putative phosphaturic substrate FGF-23 in osteoblasts, thus contributing to defective mineralization as has been suggested for other disorders including XLH and McCune Albright (54,60,65,66).

Other proteins that have been implicated in skeletal mineralization are OPN, OCN and MGP. It is unlikely that the modest down-regulation of OPN or increase in OCN would have inhibited matrix mineralization in FGFR3–/– mice, given the normal bone phenotype of OPN–/– mice (67) and the osteopetrotic phenotype of OCN–/– mice (68). In contrast, the observed increase in MGP, which is a potent inhibitor of tissue mineralization (69,70), could have contributed to the excessive osteoid and the decrease in calcein labelling seen in the FGFR3–/– mice. Compensatory signalling through FGFR1 or FGFR2 in the FGFR3–/– mice could explain the inappropriate increase in MMP13, which is upregulated by FGF2 in mature osteoblasts (71). This would in turn result in increased cleavage of type I collagen and defective mineral deposition.

In summary, we have demonstrated a role for FGFR3 signalling in post natal bone growth by showing that its absence resulted in reduced thickness of cortical bone and defective mineralization of endo-cortical and trabecular bone. From the foregoing discussion it is apparent that these defects could be a direct consequence of FGFR3 deficiency or due to alterations in the signalling pathways linked to other FGF receptors.


   MATERIALS AND METHODS
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 RT-PCR analysis of molecular...
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Mice
All mouse procedures were performed in accordance with McGill University guidelines, which are set by the Canadian Council on Animal Care. To improve the long-term viability of homozygous null offspring, mice heterozygous for inactivation of FGFR3 were bred for 10 generations onto a C3H background at McGill University. Although the skeletal phenotype of C3H FGFR3–/– mice maintained on a standard diet containing 0.97% calcium and 0.85% phosphate was the same as that of the parental Bl6 strain at Washington University, they were more robust. Male FGFR3+/+ and FGFR3–/– mice were sacrificed by exsanguination at 2 or 4 months of age for phenotypic and histologic analyses and at 1–1.5 months of age for harvesting bone marrow stromal cells for molecular biochemical analyses. Mice heterozygous and homozygous for FGFR3 gene disruption were identified using Southern blot analysis as described previously (21).

Radiologic imaging and biomechanics
A Faxitron MX20, equipped with an FPX-2 Imaging system (Dalsa Medoptics, Waterloo, Ontario, Canada) was used to obtain radiographic images and a Lunar PixiMUS 1.46 (GE-Lunar, Madison, WI, USA) to determine BMD. Morphometric analyses were performed directly on freshly dissected bones, which were then used for testing the biomechanical strength using a 3-point bending test performed at room temperature (72,73) on a Mach-1 A-300.100 (Biosensing Technologies, Montreal, Canada). Femurs were centered anterior side up on the rounded supports placed 5 mm apart so that the actuator, moving at 500 µm/s, made contact with the mid shaft of the bone. The ultimate force and displacement were determined directly from force–deformation curves and the stiffness was assessed as the slope of the linear portion of the force–deformation curve.

Micro-computed tomography
The left femur of 4-month-old mice was dissected free of soft tissue, fixed overnight in 70% ethanol and the distal metaphysis scanned with a Skyscan 1072 microCT instrument (Skyscan, Antwerp, Belgium). Image acquisition was performed at 100 kV and 98 µA, with a 0.9° rotation between frames. Two-dimensional images were used to generate three-dimensional reconstructions and to calculate morphometric parameters with the three-dimensional Creator software supplied with the instrument.


   Histomorphometry
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RT-PCR analysis of molecular...
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Mice were given intra-peritoneal injections of 30 mg/kg aqueous tetracycline at 9 days and 3 days prior to sacrifice. After micro CT analysis the left femur was embedded in polymethylmethacrylate (MMA) and 3 µm sections cut on a modified Leica RM 2155 rotary microtome (Leica Microsystems, Richmond Hill, Ontario, Canada). Fluoresecence images were captured using a Leica DMR microscope equipped with a Retiga 1300 camera (Qimaging, Burnaby, British Columbia, Canada) and histomorphometric data obtained using Bioquant Nova Prime image analysis software (Bioquant Image Analysis Corp., Nashville, TN, USA).

Histochemistry and immunohistochemistry
The right femur and tibia of the same 4 month old mice were dissected free of soft tissue, fixed for 16 h in 4% paraformaldehyde (PF) at 4°C and rinsed in PBS. The femur was embedded in a mixture of MMA and glycolmethacrylate (GMA) and 2 µm sections stained with 5% silver nitrate for 30 min under UV light and with 0.2% toluidine blue for 1 min to identify mineral and osteoid respectively. After fixing in 4% PF the tibiae were decalcified in 4.13% EDTA for 14 days at 4°C before embedding in paraffin as described previously (74).

Staining for alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) enzyme activity was carried out at 37°C in a Coplin jar placed in a waterbath. Sections were pre-incubated with aqueous 20% sucrose for 1 h, rinsed and incubated for 60 min in 50 ml of 200 mM Tris–maleate buffer (200 mM Tris, 200 mM malic acid, pH to 9.0 with NaOH) containing 5 mg napthol AS-TR phosphate in 0.5 ml N,N-dimethylformamide and 40 mg Fast Blue RR salt. Sections were rinsed three times for 10 min each with distilled water before incubating for 30 min in 50 ml of solution containing 46 ml 100 mM acetate buffer (100 ml 0.2 M acetic acid, 315 ml of 0.2 M sodium acetate and 415 ml distilled water, pH 5.0), 2 ml of fresh 4% sodium nitrite in distilled water, 2 ml of 4% pararosaniline HCl solution (1 g pararosaniline, 20 ml distilled water, 5 ml concentrated HCl warmed to 37°C and filtered), 16 mg of naphthol AS-TR phosphate in 2 ml N,N-dimethylformamide and 115 mg tartrate (Na2C4H4O6·2H2O). Sections were rinsed three times for 10 min each with distilled water, counterstained with 0.4% methyl green (Vector Laboratories Inc.) and mounted in an aqueous medium.

Immunohistochemical analyses of markers of osteoblast differentiation were performed on 4 µm sections of paraffin embedded tissue. Primary antisera were used at the indicated dilutions: type I collagen 1 : 500 (Southern Biotechnology Associates Inc., Birmingham, AL, USA); osteocalcin 1 : 200 (Biomedical Technologies Inc., Stoughton, MA, USA) and MMP13 (Chemicon International Inc., Temecula, CA, USA). Briefly, de-waxed sections were treated with 1% bovine testicular hyaluronidase (Sigma Aldrich) for 30 min at room temperature before incubating with primary antisera overnight at 4°C. Pre-immune serum was substituted for the primary antibody as a control. After washing twice with 0.1% BSA in PBS sections were incubated with biotinylated goat anti-rabbit secondary antibody (Sigma Aldrich) before incubating with the Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, Canada) and treating with the peroxidase substrate kit (Vector Laboratories Inc.). After washing with distilled water, the sections were counter-stained with 0.4% methyl green and mounted in Permount.

Biochemistry
Spot urine samples were obtained on mice before exsanguination at the time of sacrifice and the samples analysed using routine automated techniques for Ca, PO4 and ALP (serum only) at the Rodent Diagnostics Laboratory, McIntyre Medical Sciences Building, Montréal. A commercial immunoradiometric assay was used to determine serum PTH (Immunotopics Inc., San Clemente, CA, USA) and radioimmunoassays to determine 25(OH)D and 1,25(OH)2D (ImmunoDiagnostic Systems Ltd, UK)

Bone marrow stromal cell cultures
Bone marrow was flushed from the tibia and femora of 1.5-month-old mice under aseptic conditions using DMEM (Life Technologies Inc, Gaithersberg, MD, USA) supplemented with 10% FBS (Wisent Inc., St Bruno, Quebec, USA), antibiotics (Life Technologies), 50 µg/ml ascorbic acid, 10 mM ß-glycerophosphate and 10–8 M dexamethasone (all from Sigma Aldrich). Single cell suspensions were plated at a density of 106 in 60 mm2 dishes and left to adhere for 3 days when non-adherent cells were flushed away and the cultures entered day 1 of assay. Adherent cells were maintained from 3 to 18 days with media changes every 3 days. Proliferation and viability were assessed on day 3 using an MTT assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Differentiation was assessed at days 3, 6 and 18 by fixing cultures in 4% paraformaldehyde, staining for ALP as described previously (75) and by treating for 30 min with 5% silver nitrate to detect mineral deposits.


   RT–PCR analysis of molecular markers in stromal cell RNA
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 RT-PCR analysis of molecular...
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Stromal cells isolated from the long bones of three wild-type mice and three FGFR3–/– mice were plated in 35 mm dishes (six/mouse), three of which were harvested after 6 days and three after 18 days of culture. Total RNA was isolated using Trizol reagent according to the manufacturers instructions (Invitrogen/Life Technologies) and pooled from the three replicate dishes/mouse, giving a total of three independent samples of RNA for wild-type and mutant mice at each time point. A 1 µg aliquot of total RNA was subjected to reverse transcription using Superscript II (Invitrogen). PCR reactions (50 µl) contained 5 µl of 10x reaction buffer, 1 µl of 25 pmol of each primer, 1 µl of 10 mM of dNTPs mixture, 1 U of Taq polymerase, 3 µl of 25 mM MgCl2, 2 µl of RT-DNA and sterile distilled water. Reactions were initially performed with denaturation at 94°C for 5 min, extension at 72°C for 1 min and the annealing temperatures and number of cycles published in the literature. To determine the optimal conditions for each set of primers we established linear curves using different annealing temperatures and removed products after 26–40 cycles at two-cycle increments. The number of cycles at which the plateau effect occurred was thus determined individually for each target sequence, including GAPDH. Amplification products were quantitated using scanning densitometry and the values for the genes of interest normalized to those for GAPDH in the same sample. Primer sequences for FGFR3 (46), FGFR2 (46), FGFR1 (46), PTHrP (76), PTH1R (76), IHH (63), type I collagen (coll I) (77), osteopontin (OPN) (78), osteocalcin (OCN) (79), matrix GLA protein (MGP) (80) and GAPDH (81), as well as the conditions used, are shown in Table 6.


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  		Table 6. Primer sequences and reference information
 
Computer-assisted image analysis
The images shown in Figures 4–6 were captured with a Retiga 1300 camera and quantitative analyses performed with Adobe Photoshop. The histogram function was used to record the total number of pixels and average pixel luminosity, which were reported as the integrated optical density (OD). Data are presented as the mean±SD and statistical comparisons made using the Student's t-test, with a probability of P<0.05 being considered significant.


   ACKNOWLEDGEMENTS
 
This work was supported by grants to J.E.H. from the Canadian Institutes of Health Research (CIHR), the Arthritis Society of Canada (TAS) and from the Canadian Arthritis Network Centres of Excellence (CAN). G.V.-F., D.D. and H.V.-C. are recipients of doctoral training awards from the CIHR Strategic Training program, the Fonds de la recherche en santé du Québec and the MUHC Research Institute respectively. S.C. was supported by a Challenge Summer Studentship from Human Resources Development Canada and J.E.H. is a Chercheur Boursier Junior II of the Fonds de la recherche en santé du Québec. D.M.O. was supported by National Institute of Health, grant HD39952. The authors wish to acknowledge the staff of the Centre for Bone and Periodontal Research (www.bonecentre.ca) for their excellent technical assistance.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Centre for Bone and Periodontal Research, Royal Victoria Hospital, Room M11.41, 687 Pine Ave West, Montreal, QC, Canada H3A 1A1. Email: janet.henderson{at}mcgill.ca


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 MATERIALS AND METHODS
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