Human Molecular Genetics, 2000, Vol. 9, No. 2 249-258
© 2000 Oxford University Press
Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3G380R transgenic mice
1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel, 2ProChon Biotech Ltd, Kiryat Weizmann, Science Park, Rehovot 76114, Israel, 3Department of Clinical Biochemistry and 4Department of Oral Biology, Goldschleger School of Dental Medicine, Tel Aviv University, Ramat Aviv 69978, Israel
Received 13 September 1999; Revised and Accepted 15 November 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Achondroplasia is the most common genetic form of osteochondrodysplasia and short-limbed dwarfism, with an estimated frequency of 1 in 20 000 births. The disorder is transmitted in an autosomal dominant fashion with complete penetrance, although >80% of the cases arise from spontaneous mutations (1,2). The clinical features of heterozygous achondroplasia are very consistent among patients, and include proximal shortening of the extremities, depressed nasal bridge, frontal bossing, narrowing of the spinal column, narrow thorax and relative macrocephaly (3,4). Morphometric examination revealed that the growth plates are shorter than normal and that the shortening is greater in homozygous than in heterozygous cases, suggesting a gene dosage effect (5). The intercolumnar matrix is more abundant and foci of vascularization and transverse tunneling of the cartilage (ingrowth of blood vessels) are frequently observed (6).
Achondroplasia was recently shown to be caused by a point mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR-3) (7). FGFR-3 is a member of a family of four related genes encoding transmembrane receptor tyrosine kinases (8,9). They are all phosphorylated on ligand binding to activate multiple intracellular signal transduction pathways responsible for cell migration, survival, proliferation or differentiation. The FGFs and their receptors play a crucial role in tissue morphogenesis and angiogenesis. Downstream signal transduction of FGFRs is mediated primarily through the activation of the Ras-dependent and STAT signaling pathways, which result in stimulation and inhibition of mitogenesis, respectively (10,11). It was suggested that the transmembrane mutation in FGFR-3 leads to constitutive receptor activation and gain of function (12,13).
Most of the achondroplasia patients show either a G
A transition or a GC transversion, changing the codon for Gly380 (GGG) to Arg (AGG or CGG) within the receptor transmembrane domain. This G1138 nucleotide has been described as the most mutable nucleotide to date in the human genome (14). In rare cases, achondroplasia was described to result from other mutations in FGFR-3. These include a change of Gly375 to Cys (15,16) outside the transmembrane domain, and of Gly346 to Glu within the region linking the transmembrane domain with the third immunoglobulin-like domain (4).
Clinical similarities had already suggested that achondroplasia is part of a continuous spectrum of diseases that are all due to mutations in FGFR-3 and share a common defect (17). They are characterized by varying degrees of skeletal deformities, typically with disproportion between the length of the trunk and the limbs (18). On the mild side of the spectrum is hypochondroplasia, a condition associated with moderate, but variable, disproportionate shortness of limbs. On the other side of the spectrum is thanatophoric dysplasia (TD), a lethal condition that clinically resembles the lethal phenotype of homozygous achondroplasia patients (19). Other mutations in FGFR-3, as well as in other FGFRs (e.g. FGFR-1 and -2), can result in distinct skeletal and cranial malformation syndromes, which were reviewed recently (20,21).
In this report, we describe a mouse model for human achondroplasia in which the mutated human FGFR-3 cDNA (Gly380Arg) was introduced into the mouse genome under the control of the mouse FGFR-3 promoter. This results in both macroscopic and microscopic phenotypes that are remarkably similar to those of human achondroplasia cases. The mechanism by which the mutant receptor may hamper normal growth plate physiology and its implications for the development of future potential therapy are discussed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of hFGFR-3G380R transgenic mice
The tissue-specific expression pattern of ß-galactosidase driven by the FGFR-3 transcriptional control region in tissue culture cells and in transgenic mice has shown that the regulatory elements responsible for the normal expression pattern of FGFR-3 (22,23) are located in the first 5' non-coding exon and adjacent 165 bp of the upstream promoter region (O. Segev, V. Rosen, G. Levi, P. Charnay, E. Monsonego-Ornan, A. Zanzuri and A. Yayon, manuscript in preparation). McEwen and Ornitz (24) have also demonstrated that the sequence between –220 and +609 is sufficient to promote expression of a reporter gene in transgenic mice. The human FGFR-3 cDNA, containing the Gly380
Arg mutation, was therefore ligated to the mouse FGFR-3 promoter and first non-coding exon [–2311 to +156; according to McEwen and Ornitz (24)] (Fig. 1a) and the resulting construct was injected into fertilized mouse eggs to generate hFGFR-3G380R transgenic mice. Southern blots or polymerase chain reaction (PCR) of genomic DNA detected the human mutated FGFR-3 transgene in seven founder mice. Figure 1b illustrates the Southern analysis of all seven lines (1–7) and PCR analysis of line 7.
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Characterization of hFGFR-3G380R transgenic mice
Three of the founders (lines 1, 4 and 7) showed similar features, which included a short snout, a slightly larger skull, a narrow thorax and proximal limb shortening (Fig. 2a). In all other lines, the transgene was not expressed. However, the severity of this phenotype varied somewhat among different individuals of the same line. One of these lines (line 7), in which the transgene was found to be of low copy number at a single integration site, was chosen for further analysis. As determined by reverse transcription (RT)–PCR, the human transgene is expressed in chondrocytes derived from 16-day-old transgenic embryo limbs (Fig. 1c), brain, lungs and liver (data not shown), although at a lower expression level, as inspected by semi-quantitative PCR, than that of the endogenous mouse FGFR-3 (data not shown). Furthermore, in situ hybridization analysis using the antisense (AS) strand of the human FGFR-3 cDNA as a probe showed no misexpression or intense overexpression of the transgene in growth plate sections of the hFGFR-3G380R transgenic mice (Fig. 1d). Normal upregulation of FGFR-3 was detected in the lower proliferative and upper hypertrophic zones, with downregulation in the lower hypertrophic region. One should note, however, that overall fewer cells expressing FGFR-3 are found in the growth plates of the transgenic mice compared with their normal littermates.
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Mating between two transgenic mice failed to produce homozygotes. Although some died in mid-gestation, most embryos survived gestation but died within minutes after birth, probably from respiratory insufficiency. The homozygous newborns whose Southern analysis is presented in Figure 1b, lane 8, exhibited extremely short and wide extremities and underdeveloped snouts, a large skull that is more dome shaped than that of the hemizygotes, and a very small thorax (Fig. 2b). Except for skeletal abnormalities, autopsy showed no obvious gross morphological abnormalities in either the hemizygous or homozygous mice.
Skeletal abnormalities
FGFR-3-deficient mice show endochondral bone overgrowth (25,26), whereas hFGFR-3G380R mice display inhibition of bone growth. A comparison of skeletons of age-matched individuals of FGFR-3 null, normal and hFGFR-3G380R mice showed a gradual progression of skeletal features from FGFR-3 null mice to hFGFR-3G380R transgenic mice. (i) Mild shortening and broadening, in particular at the proximal (rhizomelic) segments of long bones, can be detected when comparing hFGFR-3G380R heterozygous mice with wild-type mice. A much more severe endochondral growth retardation, however, can be seen in the homozygous mice (Fig. 3d and e). A similar pattern of gradual growth retardation is observed when comparing the rib cage of the above four mice. The tubular bones become shorter and thicker, and the rib cage grows smaller and narrower (Fig. 3c). (ii) Bowing of limb bones is observed only in homozygotes (especially the tibia, fibula, radius and ulna) (Fig. 3e). (iii) The largest calvaria can be seen in the hFGFR-3G380R homozygous mice and smaller calvaria in the FGFR-3 null mice (Fig. 3a). Smaller facial bones, skull base and foramen magnum have been detected mainly in the homozygous mice including, as a result, tongue protrusion (Figs 2b and 3b). (iv) Closing of the angle at the base of the cranium is observed and is more pronounced in the hFGFR-3G380R homozygous mice (90° in homozygotes instead of 120° in normal mice) (Fig. 3a and b). (v) A relatively normal trunk size was observed for all the hemizygotes, with narrowing of the spinal column and platyspondyly, which is more severe in the homozygotes (Figs 2 and 3c). (vi) The pedicles and iliac wings are shorter and thicker with flat acetabular roofs in homozygotes (Fig. 3e). The above phenotypes are similar in many aspects to the skeletal deformities that were observed in human achondroplasia (18,21,27–30) and are summarized in Table 1.
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Measurements of long bones from hFGFR-3G380R mice and their non-transgenic littermates following alizarin red staining showed 16% shortening of the femur and 9% shortening of the tibia in hemizygous mice, and 38% shortening of the femur with 23% shortening of the tibia in homozygotes (Table 2). Similar degrees of shortening were detected using radiography of 3- and 5-month-old hemizygous mice (data not shown; see also Fig. 2a). It is interesting to note that we could not detect any significant difference in body weight between transgenic newborns and their normal littermates. Normalization of the bone lengths with body weight shows continued significant differences between transgenic and control mice for bone length per gram of body weight. In addition, all homozygous transgenic mice exhibited a marked mid-face hypoplasia with direct correlation to reduction in long bone length, suggesting that the chondrodysplasia in hFGFR-3G380R mice is not due to gestational runting, but to a delay in endochondral bone formation leading to disproportional dwarfism.
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Analysis of growth plates
Histological analysis of proximal tibia and distal femoral growth plates from 1-day-old hFGFR-3G380R mice (Fig. 4a and b) revealed shorter chondrocyte columns with less proliferative and hypertrophic cells, and some irregularly arranged chondrocytes along the columns. The growth plates of distal bones were less affected. Age-matched growth plates from homozygous newborns (Fig. 4c) were markedly shorter and had a greater circumference with a complete failure of columnization. Adequate bars of calcified chondrocytes failed to form and ossification was sparse and irregular (Fig. 4c). Foci of subchondral ossification extending into the growth plate separating the proliferating cells from the hypertrophic ones were observed in several sections from homozygous transgenic mice (data not shown). These lesions resemble the ‘ossification tufts’ that are mineralization extensions of the subchondral bone sometimes associated with achondroplasia and TD patients (6,31). At later stages, there was a marked reduction in the column size of hemizygous growth plates due to less proliferating and hypertrophic chondrocytes (Fig. 4d and e). Foci of vascularization and transverse tunneling of the cartilage were also found in the growth plate of some of our hemizygous mice (Fig. 4g), whereas no vascularization was ever apparent in the growth plates of wild-type mice (Fig. 4f).
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Since fewer cells per column were detected, we speculated that in addition to other processes this might have resulted from a decrease in the rates of chondrocyte proliferation. To test this possibility, we stained the proximal tibia growth plate of 1-week-old mice with an antibody to the proliferating cell nuclear antigen (PCNA). Growth plates isolated from 1-week-old hFGFR-3G380R mice exhibited fewer PCNA-expressing cells than those of the corresponding wild-type control mice (Fig. 4h and i). A comparison of 2000 mm2 fields from the growth plates of 1-week-old mice indicated that 22.4 ± 4 (n = 7) PCNA-labeled nuclei were counted per field in normal growth plates relative to 9.7 ± 3.14 in the transgenic mice. Quantitation of PCNA-expressing cells in growth plates of older mice reflected the same trend as mentioned above.
Since collagen type X is strongly expressed in hypertrophic chondrocytes and used as a marker for these cells, we stained epiphysis-derived growth plates with an anti-collagen type X antibody. In proximal tibia growth plates of 1-week-old hFGFR-3G380R mice (Fig. 4k), a shorter expression zone was stained relative to that of an age-matched control section (Fig. 4j). The decrease in collagen type X-expressing cells was found to be proportional to the decrease in size of the hypertrophic zone.
To assess further the involvement of hFGFR-3G380R in chondrocyte proliferation and maturation, we analyzed the proximal tibia growth plates of 15-day-old embryos (E15). At this embryonic stage, the entire tibial growth plate of the mutant mice was significantly diminished in size (Fig. 5a and b). At this stage, the reserved and proliferative zones are much smaller and hypertrophic chondrocytes are absent in homozygous transgenic embryos. Histologically, growth inhibition could be detected very early in limb development, already at the stage of early mesenchymal condensations (E12; data not shown). However, severe skeletal abnormalities could be detected only from day 15 of prenatal development onwards. The above findings suggest that the delay in chondrocyte maturation is not a property of growth plate chondrocytes only, but rather an effect of hFGFR-3G380R expression in all the cells that express the receptor, including early chondrifying mesenchymal cells.
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Later in development, these transgenic mice exhibit a marked delay in chondrocyte terminal differentiation evident within the secondary ossification centers of the proximal femur (Fig. 5c and d). In 2-month-old mutant mice, replacement of cartilage by bone and the vascular invasion within these secondary ossification centers had not yet taken place. This is in contrast to control animals, where ossification at these regions was clearly visible at this stage of development. This delay in mineralization, involving the appendicular skeleton, is also illustrated by alizarin red S staining. Although the small bones of the distal extremities in 17-day-old normal embryos are well stained, there is no staining in the corresponding bones of the hFGFR-3G380R embryos (Fig. 5e and f).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Longitudinal bone growth involves the coordination and precise balance between chondrocyte proliferation, cartilage matrix production as well as mineralization, hypertrophy and vascular invasion of the lacuna of terminally hypertrophied chondrocytes within the developing growth plates. The onset and rates of the above cellular activities vary among individuals and among various bones of one individual. These activities are under the influence of a variety of hormonal (growth, thyroid, corticosteroids and parathyroid) and local factors (insulin-like growth factors, bone morphogenic proteins and FGFs) (32) whose relative concentrations, sites and sequence of appearance vary during development. It is this tightly coordinated process, which is readily disturbed by any alteration in gene expression or function, that makes the growth plate a most sensitive organ to any such insult. Point mutations in FGFR-3, as in human achondroplasia, although expressed in many tissues, have in accordance a marked selective effect on the structure and function of the epiphyseal growth plates. It was, therefore, intriguing to find that mice expressing the human achondroplasia mutant FGFR-3 under its normal in vivo transcriptional control share such a remarkably similar phenotype with affected human individuals.
Assessing the phenotype of the hFGFR-3G380R transgenic mice, we noticed that the micromelia produced by this mutation is: (i) restricted to bones that arise by endochondral ossification; (ii) enhanced in proximal long bones compared with distal ones; and (iii) does not have a significant effect on the trunk or the calvaria (which is relatively large). These phenotypes were found in all the hFGFR-3G380R mice, although in varying degrees that appear to correlate with the dose and level of expression of the transgene (hemizygotes or homozygotes for the transgene). Assuming that we can compare between species, the hFGFR-3G380R transgenic mice phenotypes were found to exhibit many features of human achondroplasia, as summarized in Table 1. The mild heterogeneity in the severity of phenotypes of these transgenic mice, similar to that observed in human achondroplasia patients, may reflect the degree of FGFR-3 activation, as well as that of other genes which either directly modulate FGFR-3 activity or independently affect endochondral ossification. Differences in expression of FGFs and downstream effectors, for example, can ultimately determine the severity of the phenotype (33). Evidently, high expression of the mutated transgene (homozygous mice) resulted in death soon after birth, apparently due to respiratory failure, similar to homozygous human achondroplasia newborns. In both cases, the short, capped ribs and overall narrow rib cage are believed to be the cause of the respiratory insufficiency and ultimate death. This may be the reason why no other transgenic lines expressing higher FGFR-3 levels were obtained.
Recently, two mouse models displaying a dwarf phenotype and having the same mutation (G380R) were generated. In both, the mutation was introduced into the murine FGFR-3 gene, where it was either targeted to the mouse FGFR-3 locus (34) or driven by the collagen II promoter (35). In these mice, in contrast to the human FGFR-3G380R mice, the dwarf phenotype was displayed only after birth, whereas embryogenesis seemed to proceed as normal. In addition, there was no obvious disproportional, rhizomelic shortening, which is so characteristic of human achondroplasia. In the mice where the mutant FGFR-3 was driven by the collagen II promoter, the gene which was misexpressed in the growth plate and other cartilagenous tissues led to specific defects, such as in vertebra, that are not usually observed in human achondroplasia patients. The histological data presented herein are also consistent with the growth plate irregularities observed in human achondroplasia, including shorter and less organized columns. It is not clear why our transgenic animals, as opposed to the other mouse models, express the achondroplasia phenotype in utero like in humans. One possible, though highly speculative, explanation could be the expression of the reported mouse FGFR-3 pseudogene (
FGFR-3), which was mapped to chromosome 1H4-6 in an antisense orientation close to a heterologous promoter (36). The FGFR-3 mRNA, showing high homology to the mouse gene but not to the human gene, was found to be expressed only in fetal tissues, where it may specifically block the manual endogenous FGFR3 gene transcription and translation. This may suggest that the embryonic phenotype observed in our model is derived from the human transcript, which is not blocked by the mouse FGFR-3 mRNA.
The biological basis of all three human disorders (TD, achondroplasia and hypochondroplasia) seems to involve a specific defect in endochondral ossification and longitudinal bone growth. Moreover, the overgrowth of the skeleton seen in the FGFR-3 null mice (25,26) strongly suggests that FGFR-3 is a negative regulator of bone growth. The question arises as to how the above mutations in FGFR-3 influence the rate and timing of the various processes involved in endochondral ossification. Our results indicate that at as early as 12 days of prenatal development there is inhibition of endochondral ossification. The tibial growth plate of a 15-day-old embryo, where chondrocyte proliferation is prominent, is markedly diminished in size, suggesting a direct effect on cell proliferation. These less actively growing cells also seem to accumulate excessive amounts of glycogen (data not shown), as found in human achondroplasia, which can influence both cellular metabolism and matrix glycosaminoglycan deposition (37). In addition, at this stage no hypertrophic cells can be seen in the mutant mouse sections, suggesting a role for FGFR-3 in chondrocyte maturation, which is severely delayed by the G380R mutation. This is further supported by the observation that in 2-month-old mutant mice the secondary ossification center of the proximal femur epiphysis is not yet developed, in contrast to age-matched control mice where ossified bone already exists. Interestingly, mice targeted to disrupt the gene for the parathyroid hormone-related peptide (PTHrP) appear phenotypically similar to the homozygous hFGFR-3G380R mice (38). Although both mice share a severe shortage in chondrocyte columns, it is most likely through different pathways, as PTHrP apparently increases chondrocyte proliferation and slows differentiation and endochondral bone formation. It is possible that FGFR-3 and PTHrP control chondrocyte proliferation through opposite signaling pathways, and the maturation process is similarly influenced by both. An intense and prolonged expression of a potentially overactive FGFR-3 (39) throughout the hypertrophic phase may contribute to this specific delay in chondrocyte maturation and terminal differentiation in achondroplasia (40) and TD (41). It should be emphasized that although mutant receptor protein levels and stability were both found to be enhanced, the transgene RNA levels were not.
Interestingly, blood vessel invasion into the growth plate, which has been observed in human achondroplasia, was also found in our mouse model. One possible mechanism may involve changes in the extracellular matrix secreted by the arrested hypertrophic chondrocytes. We have indeed observed significant changes in matrix deposition throughout the growth plate of the mutant mice. We have also observed a significant increase in TRAP-positive osteoclast-like cells (data not shown), which are responsible for mineralized matrix remodeling and were recently shown to be involved in the vascularization of the chondro-osseus region due to the local release of matrix metalloproteinases (42). One can speculate that changes in the composition of the extracellular matrix, such as an increase in angiogenic factors such as FGFs (8,11,43), vascular endothelial growth factors (44) and co-factors such as sulfated glycosaminoglycans (45,46) and metalloproteinases (42), or alternatively a decrease in angiogenesis inhibitors, highly abundant in mature cartilage (47), can drastically affect local blood vessel formation. Such vascularization within a region that is normally avascular may severely affect growth plate physiology due to enhanced turnover and consequent changes in this tightly controlled microenvironment. As for mineralized extensions of the subchondral bone, these lesions resemble the ossification tufts seen in human homozygous achondroplasia growth plates within the upper hypertrophic region.
Finally, in this study we have produced a mouse model for achondroplasia that correlates highly with the human disorder and can be used to investigate the nature of this type of chondrodysplasia at the molecular, biochemical and anatomical levels, as well as for the development of therapeutic strategies with the potential of relieving the achondroplasia phenotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic mice
A 3.5 kb human FGFR-3 cDNA kindly provided by Dr M. Hayman (48) was used to generate the G380R mutation (40). The mutated cDNA BamHI fragment was inserted downstream of a BamHI–NotI 2.467 kb segment of the mouse FGFR-3 promoter region and first exon [nucleotides –2311 to +156; according to McEwen and Ornitz (24)] (Fig. 1a). The assembled transgene was then linearized using PvuI, microinjected into the pronuclei of CB6-F1 fertilized eggs, and resultant hFGFR-3G380R transgenic mice were identified by PCR and Southern blot analysis. Genomic tail DNA samples were digested with EcoRI (an EcoRI site was introduced 9 bp upstream of the G380R mutation) or HindIII restriction enzymes, and then probed with an EcoRI–EcoRI 1.2 kb fragment derived from the human FGFR-3 cDNA sequence. For PCR analysis, the human FGFR-3 allele was detected using a sense oligonucleotide (5'-CCTGCGTCGTGGAGAAC-3') and an antisense oligonucleotide (5'-GGACGCGTTGGACTCCAG-3'). This primer pair is specific to the human sequence and amplifies a 611 bp fragment.
RNA preparation and RT–PCR
Total RNA was extracted from limbs of 16-day-old embryos with TRI reagent (Sigma, St Louis, MO). Poly(A)+ RNA was separated from total RNA by magnetic beads using Dynabeads (Dynal, Norway). All procedures were performed according to the manufacturer’s protocols. A total of 300 ng of poly(A)+ RNA was subjected to cDNA synthesis using Expand RT (Boehringer Mannheim, Mannheim, Germany), as recommended by the supplier. A portion (10%) of this reaction was taken for PCR amplification using Taq DNA polymerase (Takara, Japan). PCR was performed for 40 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. Human FGFR-3 was detected using primers as described above. The ß-actin RT–PCR fragment was detected by a sense primer (5'-TCTACAATGAGCTGCGTGTG-3') and an antisense primer (5'-GCCGTGGTGGTGAAGCTGTA-3'). This primer pair amplifies a 343 bp fragment of the ß-actin mRNA.
Histology, immunohistochemistry and immunofluorescence
Isolated bones were fixed in 4% paraformaldehyde, or 4% formalin pH 7.4 containing cetylpyridinium chloride (0.5%), decalcified, if necessary, in EDTA, dehydrated with gradient ethanol, and embedded in paraffin. Sections (5 mm) were cut and stained with Mayer’s hematoxylin–eosin, Masson’s trichrome and with alcian blue at pH 2.5 as described (49). A polyclonal rabbit anti-mouse collagen type X antibody (dilution 1:100) was generously provided by W.A. Horton (Oregon Health Sciences University, Portland, OR) and a pre-diluted mouse anti-PCNA was purchased from Zymed (South San Francisco, CA). For immunofluorescence, Vectastain Universal ABC-peroxidase kit (Vector, Burlingame, CA) was used according to the manufacturer’s recommendations. Specific bound peroxidase was visualized by incubation with a vector VIP substrate system (Vector) and the reaction was controlled under the microscope. Slides were counterstained with methyl green (Vector) and mounted with Lemonvitrex (Carlo Erba, Italy). Negative controls were obtained by substituting the specific antibody with phosphate-buffered saline or normal serum. An Olympus BX60 microscope was used for reflected light fluorescence and transmitted light examination.
In situ hybridization
In situ hybridization was performed as described previously (50). In short, frozen tibiae of 1-week-old mice were sectioned and kept at –70°C until used. The FGFR-3 probe was a 384 bp Eco47III–EcoRI fragment, from nucleotide 779 to 1163 of the mutated human FGFR-3 cDNA described above. Sense and antisense probes were generated using either SP6 or T7 RNA polymerase, in the presence of [
-35S]UTP (Amersham, USA), using a Riboprobe II kit (Promega, Madison, WI). Hybridization was performed overnight at 50°C. Following hybridization, washing and RNase treatment (100 µg/ml, 37°C, 30 min), sections were dipped in Kodak NTB2 emulsion for 12–14 days before developing. The slides were counterstained with hematoxylin and viewed under a microscope (Olympus, Hamburg, Germany), using either bright-field or dark-field optics.
Clearance
For visualization of whole skeletons, mice were dissected free of skin, viscera and adipose tissue, and fixed in 95% ethanol followed by acetone. Bones were stained with 0.1% alizarin red S in 70% ethanol, followed by clearing of tissues in potassium hydroxide in glycerol as described (51).
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ACKNOWLEDGEMENTS |
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We thank Chu-Xia Deng for the FGFR-3 null mice, M. Hayman for human FGFR-3 cDNA and W. Horton for the anti-collagen type X antibodies. We are grateful to the Foundation Lopez Hidalgo for their generous support, to all members of the ProChon laboratories for their invaluable contribution and to L. Lipa for her excellent technical assistance. This work was supported in part by the Israel Academy of Sciences and Humanities. A.Y. is incumbent of the Alvin and Gertrude Levine Career Development Chair of Cancer Research.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +972 8 9342696; Fax: +972 8 9344125; Email: liyayon@wiccmail.weizmann.ac.il
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