Download Cys mutation of fibroblast growth factor receptor 3 in mouse

Published by Oxford University Press
Human Molecular Genetics, 2001, Vol. 10, No. 5
457–465
A Ser365→Cys mutation of fibroblast growth factor
receptor 3 in mouse downregulates Ihh/PTHrP signals
and causes severe achondroplasia
Lin Chen1,2, Cuiling Li1, Wenhui Qiao1, Xiaoling Xu1 and Chuxia Deng1,+
1Genetics
of Development and Disease Branch, Building 10, Room 9N105, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA and 2Daping Hospital,
Research Institute of Surgery, Daping, Chongqing 400042, PR China
Received 14 September 2000; Revised and Accepted 29 December 2000
Missense mutations in fibroblast growth factor
receptor 3 (FGFR3) result in several types of human
skeletal dysplasia, including the neonatally lethal
dwarfism known as thanatophoric dysplasia. An
engineered Ser365→Cys substitution in mouse
FGFR3, which is equivalent to a mutation associated
with thanatophoric dysplasia-I in humans, has now
been shown to cause severe dwarfism but not
neonatal death. The mutant mice exhibit shortened
limbs as a result of markedly reduced proliferation
and impaired differentiation of growth plate chondrocytes. The receptor-activating mutation also resulted
in downregulation of expression of the Indian
hedgehog (IHH) and parathyroid hormone-related
protein (PTHrP) receptor genes, both of which are
important for bone growth. Interactions between
FGFR3- and PTHrP-receptor-mediated signals during
endochondral ossification were examined with
embryonic metatarsal bones maintained in culture
under defined conditions. Consistent with the in vivo
observations, FGF2 inhibited bone growth in culture
and induced downregulation of IHH and PTHrP
receptor gene expression. Furthermore, PTHrP
partially reversed the inhibition of long bone growth
caused by activation of FGFR3; however, it impaired
the differentiation of chondrocytes in an FGFR3independent manner. These observations suggest
that FGFR3 and IHH-PTHrP signals are transmitted
by two interacting parallel pathways that mediate
both overlapping and distinct functions during endochondral ossification.
INTRODUCTION
Vertebrate long bones are formed by endochondral ossification,
which involves the production of cartilage tissue through
mesenchymal cell condensation and its subsequent replacement
by bone. During this highly coordinated process, chondrocytes
+To
undergo programmed cell proliferation, differentiation, death
and replacement by osteoblasts (1).
Endochondral ossification is regulated by many growth
factors and signaling molecules, including fibroblast growth
factor receptor 3 (FGFR3), Indian hedgehog (IHH), parathyroid
hormone-related protein (PTHrP) and its receptor (PTHrP-R),
STAT (signal transducer and activator of transcription)
proteins, and cell cycle inhibitors (2–8). FGFR3 is one of four
membrane-spanning tyrosine kinase receptors that mediate
signals of at least 21 FGFs (9,10). This receptor was first
implicated in bone growth by the observation that a Gly→Arg
missense mutation (G380R) in its transmembrane domain
results in achondroplasia, the most common form of dwarfism
in humans (11,12). Many additional mutations in various
regions of FGFR3 were subsequently shown to cause skeletal
dysplasias of varying severity, including hypochondroplasia,
severe achondroplasia with developmental delay and
acanthosis nigricans (SADDAN) and thanatophoric dysplasia
(TD) (13–16).
TD is the most severe form of dwarfism caused by FGFR3
mutations (14). Affected individuals exhibit markedly shortened limbs and generally die of respiratory failure within a few
hours after birth. The two distinct types of TD are distinguished
clinically by the presence of straight femurs and cloverleaf
skull (TD-II) or curved short femurs (TD-I). TD-II is caused by
a K650E mutation in the tyrosine kinase domain of FGFR3,
whereas several different point mutations, including R248C,
S249C, S371C, Stop807G, Stop807R and Stop807C, cause
TD-I (15,17–19). Most individuals with TD-I harbor the
R248C mutation, with the other mutations occurring less
frequently (19,20).
FGFR3 mutations examined so far result in ligand-independent
phosphorylation of the tyrosine kinase domain (5,21–23).
Furthermore, the FGFR3-TD-II mutation activates STAT
proteins both in vivo and in vitro (7). This results in upregulation
of cell cycle inhibitors that block chondrocyte proliferation and
retard bone growth, as demonstrated recently for mutations
that cause achondroplasia and TD-II (5–7). Expression of IHH
is also reduced in dwarf mice expressing activated FGFR3
(6,24), suggesting that signaling through FGFR3 negatively
regulates IHH expression.
whom correspondence should be addressed. Tel: +1 301 402 7225; Fax: +1 301 480 1135; Email: [email protected]
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Human Molecular Genetics, 2001, Vol. 10, No. 5
Given that both FGF-FGFR3 and IHH-PTHrP signals play
important roles in chondrocyte proliferation and differentiation
and that FGFR3 signaling appears to downregulate the expression
of IHH (2–4,25–27), it is possible that these two pathways
interact functionally during endochondral bone growth. To
shed light on such interactions and their role in skeletal
dysplasia, as well as generating mouse models representing
various FGFR3 mutations, we introduced an S365C mutation,
which corresponds to the TD-I-associated S371C mutation in
human FGFR3, into the mouse genome using gene targeting.
The heterozygous mutant mice were unexpectedly viable and
exhibited skeletal dysplasia similar to, but more severe than,
human achondroplasia. Unlike previously described achondroplastic mice (5,6,24,28,29), the FGFR3-S365C mutant animals
also exhibited skeletal dysplasia at embryonic stages, making
it possible to study molecular interactions in explant cultures
of embryonic bone. Using this culture system, we have demonstrated that decreased endochondral bone growth caused by
activated FGFR3 or basic FGF (bFGF) treatment is accompanied
by downregulation of IHH and PTHrP-R gene expression,
suggesting a role for IHH-PTHrP–PTHrP-R signaling in
FGFR3-associated skeletal dysplasias. Moreover, our data
suggest that FGF-FGFR3 signaling inhibits chondrocyte
proliferation through the regulation of IHH expression, and
that FGF-FGFR3 and PTHrP–PTHrP-R independently inhibit
chondrocyte differentiation.
RESULTS
The S365C mutation of FGFR3 results in viable dwarf mice
To study the effect of the S365C mutation of FGFR3 on bone
growth, we introduced the mutation into the mouse genome
(Fig. 1). Because the presence of the neo gene in intron 10
prevented normal splicing of the mutant Fgfr3 gene (data not
shown), resulting in a phenotype resembling that of FGFR3
knockout mice (3), we crossed the heterozygous mutant
animals with EIIa-Cre mice (30) to excise the neo sequence
from the germline (Fig. 1B). After removal of the neo gene, the
mutant allele was expressed at the same level as was the wildtype allele, as revealed by northern blot analysis (data not
shown). All data presented below were obtained from mice
lacking the neo gene.
At birth, mice heterozygous for the S365C mutation
(Fgfr3365/+) exhibited skulls that were slightly dome-shaped
(data not shown), which became progressively more pronounced as the mice aged (Fig. 2A). The skulls of Fgfr3365/+
mice were markedly reduced in size along the anteroposterior
axis, whereas they were slightly larger along the left–right and
dorsal–ventral axes compared with wild-type animals (Fig. 2A,
C–F). The synochondroses of mutant mice had fused prematurely and ossified, resulting in a much shorter cranial base
(Fig. 2E and F). The Fgfr3365/+ mice exhibited severe dwarfism
postnatally characterized by reduced length of the long bones,
especially of femurs and tail bones (Fig. 2A and B). Some
mutant animals manifested bowed tibias and fibulas (Fig. 2A),
impairing their movement. The Fgfr3363/+ animals also displayed longer incisors (not shown), possibly a consequence of
the shortened cranial base that disrupts normal tooth alignment. The overgrown incisors were cut weekly allowing
Figure 1. Introduction of the S365C mutation into the mouse Fgfr3 locus.
(A) Schematic representation of the targeting construct and removal of the neo
gene. The targeting vector, pFgfr3-S365C, contained the S365C mutation
(asterisk) in exon 10 of Fgfr3, a LoxPneo construct in intron 10 of Fgfr3 and the
thymidine kinase (TK) gene. (B) Removal of LoxPneo by breeding animals showing germ-line transmission of the targeted allele with EIIa-Cre mice. (C) Southern
blot analysis of G418- and FIAU-resistant ES cell clones. Of 140 clones examined by Southern blot analysis of SpeI (Sp)digested genomic DNA with a 5′
flanking probe (probe 1), three (lanes 1–3) exhibited, in addition to the wildtype fragment, a fragment of ∼9.4 kb. The targeting events were confirmed by
digestion of genomic DNA with XbaI (Xb) and EcoRV (Ev) and Southern blot
analysis with a 3′ internal probe (probe 2) (not shown) and sequencing analysis
also verified the presence of the point mutation (not shown). Probe 1, a 1.9 kb
SacII–BamHI fragment, and probe 2, a 1.65 kb SpeI–XbaI fragment, are 5′
flanking and 3′ internal to the targeting construct, respectively.
mutant mice to eat and drink normally. Under such conditions,
some mutant mice survived for up to 6 months.
We investigated whether the Fgfr3365/+ mice were fertile.
Heterozygous animals at ages of 1.5–6 months were allowed to
mate with other heterozygotes or with wild-type mice. All
Fgfr3365/+ males were infertile. Two of 10 Fgfr3365/+ females
that were mated with wild-type males each produced one litter
of offspring; subsequent mating of the same pairs of animals
failed to generate any more litters. The reduced fertility of the
mutant mice is possibly due to their smaller size or to restrictions
on their hindlimb movement, given that no abnormalities were
apparent in their testes or ovaries (not shown).
Disorganized growth plates of Fgfr3365/+ mutant mice
Histological examination of epiphyseal growth plates revealed
differences between wild-type and mutant mice that were first
apparent at embryonic day (E)17.5. Unlike the growth plates of
wild-type animals, which exhibit distinct borders between each
zone of chondrocytes (resting, proliferating, prehypertrophic
and hypertrophic chondrocytes) (Fig. 3A), growth plates of
mutant mice showed no clear boundaries between these zones
Human Molecular Genetics, 2001, Vol. 10, No. 5
Figure 2. Dwarfism of Fgfr3365/+ mice. (A) Alizarin red S and Alcian blue staining
of the skeletons of P30 wild-type (WT) and Fgfr3365/+ mice. Arrow and arrowhead
indicate the dome-shaped skull and bowed tibia of the mutant mouse, respectively.
(B) Tail length of wild-type and Fgfr3365/+ mice at various postnatal ages. Data
are means of values obtained from five mice of each genotype; SD values were
<5% of the mean. (C–F) Skulls of P30 wild-type (C and E) and Fgfr3365/+ (E and
F) mice. Dorsal (C and D) and ventral (E and F) views are shown. Arrow in (F)
indicates the prematurely fused synochondroses of the mutant compared with
WT mice (E). Bars; 22 mm (C–F).
(Fig. 3B). Chondrocyte columns (longitudinal stacks of cells)
were markedly shorter and hypertrophic chondrocytes were
reduced in number in the growth plates of all mutant mice
examined at ages between E17.5 and postnatal day (P)45
(Fig. 3B, E and G; data not shown). Chondrocytes in the
maturing zone were sometimes mingled with those in the
hypertrophic zone of younger (<P20) mutant mice (Fig. 3C–E).
Many histologically less well differentiated small spherical
chondrocytes, some of which were directly opposed to the ossification front, invaded the hypertrophic zone (Fig. 3C and D).
Conversely, prehypertrophic chondrocyte-like cells were often
observed in resting or proliferating zones in Fgfr3365/+ mice
(Fig. 3C–E). These observations suggest that the FGFR3-S365C
mutation uncouples the coordination between chondrocyte
proliferation and differentiation. The growth plates of older
(>P30) mutant animals (Fig. 3G) were much narrower than
age-matched controls (Fig. 3F) with fewer proliferating and
hypertrophic chondrocytes, indicating that their activities had
become minimal.
Reduced chondrocyte proliferation and differentiation in
Fgfr3365/+ mutant mice
Chondrocyte proliferation was examined using [3H]thymidine
labeling. [3H]thymidine labeled cells were localized primarily
in the proliferating zone of P15 wild-type mice (Fig. 4A),
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Figure 3. Histology of epiphyseal growth plates of wild-type and Fgfr3365/+ mice.
(A) Growth plate of a wild-type mouse (P9), consisting of resting (R), proliferating
(P), prehypertrophic (PH) and hypertrophic (H) zones. (B) Disorganized growth
plate of a mutant mouse (P9). (C–E) Abnormal hypertrophic chondrocyte columns
in the growth plates of mutant mice at P5, P9 and P12, respectively. Arrows in
(C) and (D) indicate prehypertrophic chondrocytes protruding into the hypertrophic
zone. Arrowheads in (C), (D) and (E) indicate prehypertrophic chondrocytes
mingled with resting zone chondrocytes. (F and G) Growth plates of older
(P40) wild-type and mutant mice, respectively. The growth plate of the mutant
is much narrower than that of the wild-type mouse. Bars, 170 µm (A and B) and
85 µm (C –G).
whereas [3H]thymidine-labeled cells in mutant animals were
fewer in number and scattered throughout the growth plate
(Fig. 4B). The labeling intensity of mutant cells was also
decreased. These observations suggest that the proliferative
capacity of mutant chondrocytes is reduced compared with that
of chondrocytes in wild-type littermates. We have previously
shown that activated Fgfr3 signals activate Stats (3–5).
Consistently, Stat1 expression in the nuclei of mutant cells was
more intense than in wild-type cells (Fig. 4C and D).
In addition to proliferating, chondrocytes in the proliferation
zone also undergo programmed differentiation into hypertrophic cells, a process characterized by a gradual increase in
cell size while they move downward into the hypertrophic
zone in the growth plate. Histologically, mutant chondrocytes
appeared significantly smaller (Figs 3B and 4B) than wild-type
cells (Figs 3A and 4A), indicating that the mutant cells were
less well differentiated. Consistently, in situ hybridization
using a probe for collagen type X gene, a specific marker for
hypertrophic chondrocytes, revealed that the zone of hypertrophic chondrocytes was much narrower in mutant growth
plates (Fig. 4E and F).
To further characterize the differentiation of mutant
chondrocytes, we examined the expression of the IHH and
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Figure 5. Effect of bFGF on the growth in culture of metatarsal bones isolated
from E17.5 wild-type mice. (A–D) Bones before and after culture for 3 days in
the absence (A and B) or presence (C and D) of bFGF (10 ng/ml), respectively.
The dark area corresponds to the zone of mineralized hypertrophic chondrocytes
(MIN), and the light areas flanking the dark region represent non-mineralized
hypertrophic chondrocytes. The length of the hypertrophic zone (HL) includes
the regions of both mineralized and unmineralized hypertrophic chondrocytes. The
length of proliferation zone (PL) is calculated as the difference between the total
length of the bone (TL) and HL divided by two. (E and F) In situ hybridization
analysis of bones cultured for 36 h in the absence or presence of bFGF with
probes specific for PTHrP-R (E) and IHH (F). (G) Quantitation of the percentage
increases in TL, HL and PL for bones cultured for 3 days in the absence (control)
or presence of bFGF. Data are means ± SD of values obtained from seven pairs
of bones.
Figure 4. [3H]thymidine labeling and in situ hybridization analyses of growth plates
of P15 wild-type and Fgfr3365/+ mice. (A and B) [3H]thymidine incorporation
(arrows) in wild-type (A) and mutant (B) mice. The labeled cells in the growth
plate of the wild-type animal indicate the proliferation zone. In contrast, those
in the mutant are fewer in number and scattered throughout the growth plate,
suggesting that most of the cells are in a quiescent state. (C and D) Immunohistochemical staining (arrows) using an antibody to Stat1 in wild-type (C) and
mutant (D) chondrocytes. (E–L) In situ hybridization analysis of growth plates
of wild-type (E, G, I and K) and mutant (F, H, J and L) mice with probes specific
for transcripts of collagen type X (ColX) (E and F), PTHrP-R (G and H) or IHH
(I and J) genes. The insets in (I) and (J) are enlarged in (K) and (L), respectively.
The expression of all three genes was reduced in the mutant mice compared
with that in wild-type animals. Bars, 100 µm (A –D, K and L) 150 µm (E and
F) and 350 µm (G –J).
PTHrP-R genes, specific markers of prehypertrophic chondrocytes. Expression of both genes was markedly downregulated,
as reflected by reduced numbers of positive cells and signal
intensity, in the mutant mice (Fig. 4G–L). Together, these
observations indicate that the FGFR3-S365C mutation impairs
chondrocyte differentiation.
Downregulation of Ihh and PTHrP-R gene expression by
bFGF in cultured bone
IHH, PTHrP and PTHrP-R contribute to a signaling pathway that
plays essential roles in regulating chondrocyte proliferation and
differentiation (25,26). The downregulation of Ihh and PTHrP-R
gene expression in the growth plates of Fgfr3365/+ mice is
consistent with previous investigations showing that Ihh
expression levels are lower in dwarf mice carrying activated
FGFR3 (6,24). However, since the zones of chondrocytes that
express Ihh and PTHrP-R in growth plates of all these mutant
strains are narrower than those of control mice, it is not clear
that the decreased expression of these genes is directly or indirectly caused by activated FGFR3. To distinguish this, we
employed an embryonic bone culture system, in which bone
growth and gene expression pattern can be monitored under
defined conditions. We showed that the bone rudiments from
E17.5 embryos had already initiated endochondral ossification,
with a dark area in the center representing the zone of mineralized
hypertrophic chondrocytes and light regions flanking the dark
area corresponding to the zone of unmineralized hypertrophic
chondrocytes (Fig. 5A). The regions extending from the light
areas to the ends of the bone represent proliferating and articular
chondrocytes. Bone growth was revealed by increased total
bone length and hypertrophic zone toward the ends of the
rudiment after incubation for 3 days (Fig. 5A and B). In the
presence of bFGF (10 ng/ml), however, the increases in the
lengths of the hypertrophic zone were greatly reduced (Fig. 5C
Human Molecular Genetics, 2001, Vol. 10, No. 5
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and D), indicating inhibition of bone growth and chondrocyte
differentiation. Quantitative analysis also revealed that the
percentage increase in total bone length was reduced by bFGF
treatment (Fig. 5G). We next determined PTHrP-R and Ihh
transcription levels by in situ hybridization at multiple time
points. Our data revealed that the abundance of PTHrP-R and
Ihh mRNAs in the bone rudiments was markedly reduced in
the presence of bFGF for 36 h (Fig. 5E and F). At this time
point, no changes in bone growth were observed. Thus,
PTHrP-R and Ihh downregulation precedes the appearance of
bone abnormality and excludes the possibility that decreased
expression of these genes is a consequence of bone dysplasia.
FGF-FGFR3 and PTHrP–PTHrP-R signals function
independently in inhibiting chondrocyte differentiation
Overexpression of PTHrP or PTHrP-R inhibits chondrocyte
differentiation (31). Conversely, mice deficient in IHH, PTHrP
or PTHrP-R exhibit premature chondrocyte hypertrophy
(4,25,26). However, the downregulation of PTHrP-R expression
observed in Fgfr3365/+ mice does not result in premature hypertrophy of chondrocytes. Alternatively, the decrease in PTHrP–
PTHrP-R signaling was not able to reverse the inhibition of
chondrocyte differentiation induced by FGF-FGFR3 signaling.
This observation suggests that FGF-FGFR3 signaling inhibits
chondrocyte differentiation independently, irrespective of
PTHrP–PTHrP-R signaling.
Therefore, we investigated whether the inhibition of
chondrocyte differentiation by PTHrP–PTHrP-R signaling is
dependent on FGF-FGFR3 signaling. Culture of bones from
wild-type embryos in the presence of PTHrP markedly inhibited
chondrocyte differentiation, as reflected in the reduced extent of
the increase in the length of the hypertrophic zone (Fig. 6A–D, O).
This result is consistent with a previous observation that
cultured bones from PTHrP knockout mice exhibit a marked
hypertrophic expansion (32).
Next, we examined bones isolated from Fgfr3365/+ mice.
Prior to culture, the mutant hypertrophic zone was much
shorter than wild-type bone (Fig. 6A and E), indicating that the
skeletal dysplasia caused by S365C mutation was apparent at
this early developmental stage. The length of the hypertrophic
zone of the mutant bone was increased by culture for 3 days,
although to a reduced extent compared with the increases
observed in wild-type bone (Fig. 6B, F and O). This observation
suggests that the S365C mutation did not completely activate
the FGF-FGFR3 signaling pathway. As with wild-type bone,
culture of bone from Fgfr3365/+ mutant mice with PTHrP markedly
reduced the extent of the increase in the length of the hypertrophic zone (Fig. 6E–H, O), again indicating inhibition of
chondrocyte differentiation by PTHrP. We also showed that
PTHrP inhibited chondrocyte differentiation in cultures of
bone isolated from FGFR3 knockout mice (Fig. 6K–O). These
data thus indicate that inhibition of chondrocyte differentiation
by PTHrP is independent of FGFR3.
PTHrP increases the length of the proliferation zone but
not the rate of chondrocyte proliferation in cultured bone
PTHrP induced increases in proliferation zone length in
cultured wild-type and mutant bones (Fig. 6A–H). In contrast,
although bFGF reduced the extent of the increase in the size of
the hypertrophic zone of wild-type bone, it had very little
Figure 6. Effects of PTHrP on the growth in culture of metatarsal bones isolated
from wild-type, Fgfr3365/+ and Fgfr3–/– mice. Genotypes and time of PTHrP
(0.1 µM) treatment were as indicated. (A–J) PTHrP inhibited the expansion
during culture of the hypertrophic zone of bones from both wild-type and mutant
mice. While PTHrP induced a slight increase in the total length of wild-type bone
(B and D), it markedly increased the total length of mutant bone (F and H).
(I and J) Histological analysis of [3H]thymidine incorporation into Fgfr3365/+
bones cultured in the absence (I) or presence (J) of PTHrP, respectively. No
significant differences in percentages of [3H]thymidine-labeled cells were
found when reviewed at higher powers (inserts). ([3H]thymidine was added 2 h
before the samples were harvested). (K–N) Bones isolated from Fgfr3–/– mice.
(O) Percentage increases in the total length (TL) and hypertrophic zone length
(HL) after culture of bones for 3 days in the absence or presence of PTHrP. Data
are means ± SD of values obtained from nine bones.
effect on the proliferation zone (Fig. 5A–D, G). Because the
chondrocyte proliferation rate was reduced in Fgfr3365/+ mice,
we examined whether the PTHrP-induced expansion of the
proliferation zone was attributable to increased chondrocyte
proliferation. However, we detected no marked difference in
[3H]thymidine incorporation between mutant bones cultured in
the absence or presence of PTHrP (Fig. 6I and J). PTHrP–
PTHrP-R signaling promotes the reversion of hypertrophic
chondrocytes to prehypertrophic and proliferating cells (33). It
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Human Molecular Genetics, 2001, Vol. 10, No. 5
is thus possible that PTHrP treatment blocked chondrocyte
differentiation and promoted reversion of hypertrophic cells to
a less well-differentiated state. This effect would not only
increase the pool of proliferating chondrocytes, but also
prolong the time spent by cells in the proliferating state,
thereby allowing them to undergo an increased number of cell
divisions (without necessarily increasing their proliferation
efficiency) before they enter the terminal differentiation
pathway.
DISCUSSION
FGF-FGFR3 and IHH-PTHrP–PTHrP-R signaling pathways
are important regulators of chondrocyte proliferation and
differentiation during endochondral ossification (24,25). On
the basis of the observation that IHH expression is markedly
downregulated in mice that harbor activating mutations of
FGFR3, it has been suggested that FGFR3 functions upstream
of IHH and negatively regulates its expression. However, it has
remained unclear whether FGF-FGFR3 signaling and IHHPTHrP–PTHrP-R signaling interact during bone growth. We
have now provided additional evidence that activated FGFR3
signals downregulate expression of IHH, which is known to
play an essential role in promoting chondrocyte proliferation
(25). In addition, we demonstrated that FGF-FGFR3 and
PTHrP–PTHrP-R signals independently inhibit chondrocyte
differentiation. On the basis of these observations, we propose
that FGF-FGFR3 and IHH-PTHrP–PTHrP-R signals are transmitted by two integrated parallel pathways that perform both
overlapping and distinct functions during long bone growth
(Fig. 7).
FGF-FGFR3 signaling targets STATs and IHH to inhibit
chondrocyte proliferation
Mouse mutants harboring activating mutations of FGFR3
exhibit skeletal dysplasia characterized by reduced chondrocyte proliferation (5,6,24,28). It was shown that FGFR3
containing the TD-II mutation activates STAT1 in growth
plate chondrocytes of TD-II patients, leading to upregulation
of p21, a cell cycle inhibitor that blocks the G1–S transition (7).
Activation of several STAT proteins, including STAT1,
STAT5a and/or STAT5b, has also been detected in mouse
models of achondroplasia that express activated forms of
FGFR3 (5,6 and this study). Inhibition of bone growth by FGF
has also been observed both with cultured bone (34) and in
transgenic mice (35); such inhibition was not apparent with
cultured bone isolated from STAT1-deficient mice (8). These
data thus support the notion that FGF-FGFR3 signaling
inhibits bone growth through the activation of STAT proteins
(Fig. 7).
The downregulation of IHH and PTHrP-R gene expression
apparent in Fgfr3365/+ mice suggests a role for IHH and PTHrP
signals in the pathogenesis of FGFR3-associated skeletal
dysplasia. An essential role for IHH in chondrocyte proliferation
and differentiation was recently revealed by the observation
that IHH-deficient chondrocytes exhibit minimal proliferation
and premature hypertrophy (25). The expression of IHH in
prehypertrophic chondrocytes was shown to induce expression
of PTHrP in the articular perichondrium (4). The expression of
PTHrP, in turn, results in activation of PTHrP-R in prehyper-
Figure 7. Model of the relations between FGF-FGFR3 and IHH-PTHrP–PTHrP-R
signaling in endochondral bone formation. FGF-FGFR3 and IHH-PTHrP–PTHrP-R
signals are transmitted by two integrated parallel pathways that mediate both
overlapping and distinct functions during the growth of long bones. Both FGFR3
and IHH affect chondrocyte proliferation. However, FGFR3 is a negative regulator
of bone growth, whereas IHH positively regulates bone growth. Evidence
suggests that FGF-FGFR3 signaling induces activation of STAT proteins,
upregulation of the expression of cell cycle inhibitors and downregulation of
IHH expression. Both FGF-FGFR3 and PTHrP–PTHrP-R signals inhibit
chondrocyte differentiation, and both signals appear to act in a dominant and
independent manner.
trophic chondrocytes (4). The observation that expression of
PTHrP was not detected in IHH-deficient articular perichondrium (25) is consistent with this view.
However, the absence of PTHrP signaling is only partially
responsible for the skeletal phenotype of IHH-deficient mice
(25). The introduction of a constitutively active PTHrP-R into
IHH-deficient mice failed to rescue either the short-limb
dwarfism or the reduced chondrocyte proliferation apparent in
these animals, although it prevented the premature chondrocyte hypertrophy (27). These observations not only provide
further evidence for a positive role of IHH signaling in regulating chondrocyte proliferation, but also suggest that IHH
controls chondrocyte proliferation and differentiation in
PTHrP–PTHrP-R-independent and -dependent manners,
respectively.
FGF-FGFR3 and PTHrP–PTHrP-R signals inhibit
chondrocyte differentiation independently
Our data show that the reduced chondrocyte differentiation in
the growth plates of Fgfr3365/+ mice and in bFGF-treated bone
rudiments is accompanied by downregulation of PTHrP-R
gene expression. These results suggest the possibility that
reduced PTHrP-R expression is responsible for impaired
chondrocyte differentiation. However, previous data obtained
Human Molecular Genetics, 2001, Vol. 10, No. 5
from both in vivo and in vitro studies are not consistent with
this possibility since loss-of-function mutations in PTHrP or
PTHrP-R result in premature hypertrophy of chondrocytes in
mice (26,36,37), and in cultured bone rudiments (32). Thus,
the activated FGF/FGFR3 signals appear to override the effect
of reduced PTHrP signaling on chondrocyte differentiation.
We also considered the possibility that FGF-FGFR3 signaling
functions downstream of PTHrP–PTHrP-R. However, we
showed that the effect of PTHrP treatment on chondrocyte
differentiation is independent of FGFR3, irrespective of
FGFR3 mutation status. Given that both FGF-FGFR3 and
PTHrP–PTHrP-R signals are dominant, we propose that these
signals function independently in repressing chondrocyte
differentiation (Fig. 7). According to this model, the joint
action of both signals would be predicted to exert a more
marked inhibitory effect than either signal alone. Consistently,
PTHrP treatment further inhibited chondrocyte differentiation
in Fgfr3365/+ mutant bones.
Achondroplasia, not lethal dwarfism, is associated with the
S365C mutation of FGFR3 in mice
The S371C mutation in human FGFR3 results in TD-I, a
neonatally lethal skeletal dysplasia (20). However, the corresponding S365C mutation in mouse FGFR3 is not lethal,
exhibiting a markedly less severe skeletal phenotype. This
phenotypic discrepancy could be attributed to multiple factors.
First, different genetic backgrounds can give rise to markedly
different phenotypes (38,39). Although individuals with TD-I
usually die at birth, some survive for many years (16,40).
Given that humans are heterogeneous, epigenetic factors may
affect TD-I severity. Second, the severity of skeletal phenotype
is approximately proportional to the level of activation of
various FGFR3 mutants (22). We have previously shown that
the S365C mutation of FGFR3 results in a much lower level of
receptor tyrosine kinase activity than does K650E, a TD-II–
associated mutation that causes neonatal death both in humans
(5) and in mouse when it is expressed properly (41). These
observations suggest that the S365C mutation of mouse
FGFR3 is relatively weak in terms of its effect on FGFR3
activity, possibly explaining its associated mild phenotype.
Although Fgr3365/+ mice survive at birth, they exhibit a
phenotype more severe than other achondroplastic mice
generated previously in our laboratory (5,6). They manifested
an embryonic onset of skeletal dysplasia as well as bowed tibia
in older animals. They also exhibited a high postnatal
mortality, with many animals dying between 1 week and
6 months of age. All of the described TD patients that survived
after birth died before reaching 10 years of age (16,42,43). The
Fgr3365/+ mice might therefore represent a model for such
surviving individuals with severe achondroplasia. These
animals together with the other FGFR3 mutant mice created
previously represent a spectrum of skeletal dysplasia encompassing severe, intermediate and mild forms of dwarfism that
mimic various forms of human FGFR3-associated disease.
They should therefore prove useful as models for studying the
mechanisms that underlie the human diseases as well as for the
development of new therapeutic strategies.
463
MATERIALS AND METHODS
Site-directed mutagenesis and targeting vector
construction
The S365C mutation was introduced into exon 10 of the mouse
Fgfr3 gene with the use of the oligonucleotide 5′-GGAAACTGATGAGGCTGGCTGCGTGTACGCAGGCGTCCTCAGC-3′
(underline indicates the mutated nucleotide) and a standard
protocol for site-directed mutagenesis. The targeting vector
was constructed with the use of Fgfr3 genomic DNA isolated
previously (3). A 2.8 kb EcoRI–SmaI fragment of Fgfr3
genomic DNA containing the S365C mutation was inserted
into the EcoRI and BamHI sites of the pLoxpneo vector (44).
The resulting plasmid was digested with HpaI and NotI, and a
3.9 kb SmaI–XbaI fragment of Fgfr3 genomic DNA was
inserted. The resulting targeting construct, pFgfr3-S365C, is
shown in Figure 1A.
Homologous recombination in ES cells and generation of
germ-line chimeras
Mouse TC1 embryonic stem (ES) cells (3) were transfected
with NotI-digested pFgfr3-S365C. Genomic DNA from
resulting G418- and FIAU-resistant ES cell clones was
digested with restriction enzymes and subjected to Southern
blot analysis with a 5′ flanking (Fig. 1B) or a 3′ internal (not
shown) probe specific for Fgfr3. Blastocyst injection and
screening for germ-line transmission were performed
according to standard procedures.
Genotype analysis
Mice exhibiting germ-line transmission of the S365C mutation
were crossed with EIIa-Cre mice (37) in order to delete the neo
gene. Genotypes of the resulting progeny were determined by
polymerase chain reaction analysis with primers 1 (5′-CCGGGGGAAAGCTTGAAAA-3′) and 2 (5′-TGTAAAAGGGGTGGGGTGGTAG-3′), which flank the SmaI site and amplify
DNA fragments of 534 and 594 bp from wild-type and mutant
Fgfr genes, respectively. The mutant fragment is larger
because of the presence of a LoxP site.
Histology and skeletal staining
Histological sections (5 µm) were prepared from selected
tissues that had been fixed in 10% formalin, decalcified by
incubation with 15% EDTA at 4°C for 1 week, and embedded
in paraffin. Skeletal staining with Alizarin red S and Alcian
blue was performed as described (45).
In situ hybridization and [3H]thymidine labeling
In situ hybridization was performed according to standard
procedures with probes listed in the Acknowledgements
section. [3H]thymidine (Amersham Pharmacia Biotech) was
injected intraperitoneally at a dose of 20 µCi/g of body weight
and animals were killed 2 h later. Slides were dipped in emulsion (Kodak NTB-2) and exposed for 4 to 15 days before
developing.
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Human Molecular Genetics, 2001, Vol. 10, No. 5
Culture of embryonic metatarsal rudiments
Culture of metatarsal rudiments isolated from 17.5-day pregnant
mice was performed as described (32). PTHrP (0.1 µM)
(Sigma) or bFGF (10 ng/ml) (Sigma) dissolved in 4 mM HCl
was added to cultures after 16–20 h. The culture medium was
changed on the second day of culture with PTHrP or bFGF.
The total length and hypertrophic zone length were measured
with an eyepiece scale of a Leica dissecting microscope.
Changes in length were expressed as percentage increase relative to the value before treatment. Data are expressed as means
± SD, and the significance of differences was evaluated with
Student’s t-test.
ACKNOWLEDGEMENTS
We thank the Press family foundation for their generous and
continuous support to this project. We thank B. Olsen for
ColX, A. Broadus for PTHrP-R, A. McMahon for IHH in situ
probes and H. Westphal for the EIIa-cre mice. We thank S.G.
Brodie for critical reading of this manuscript.
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