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First published online July 27, 2007
doi: 10.1242/10.1242/dev.007542
,
1 Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle
upon Tyne NE1 3BZ, UK.
2 Centro de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid,
Spain.
3 Department of Human Anatomy and Embryology, Faculty of Medicine, Universidad
Complutense de Madrid, 28040 Madrid, Spain.
Authors for correspondence (e-mails:
vlruiz{at}iib.uam.es;
j.a.goodship{at}ncl.ac.uk)
Accepted 7 June 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Evc, Ihh, Hedgehog signalling, Chondrocyte, Basal body, Gli3 processing
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Although medical management of both respiratory and cardiovascular problems
has improved dramatically, there is still a significant mortality associated
with this disorder.
By positional cloning we identified two genes, EVC and
EVC2, which when mutated give rise to this condition
(Ruiz-Perez et al., 2000;
Ruiz-Perez et al., 2003).
These genes are in close proximity, with a divergent orientation; the
transcription starts sites are separated by 2,624 bp in human and by only
1,647 bp in mouse. EVC and EVC2 encode novel proteins with
putative transmembrane domains and regions of coiled-coiled structure, but on
database interrogation show no similarity with any other proteins or motifs
that give clues to their function. To determine which molecular pathways and
developmental processes are perturbed in EvC, we developed anti-Evc antibodies
to study subcellular localisation and generated a mouse model. As the majority
of mutations identified in EVC
(Tompson et al., 2007) are
predicted to cause loss of function, we ablated gene function in the
mouse.
The major features of EvC are shortening of the long bones and ribs.
Although many signalling molecules and pathways are involved in skeletal
development, Indian hedgehog (Ihh) is the master regulator
(Kronenberg, 2003). In the
growth plates of long bones, Ihh is secreted by the prehypertrophic
chondrocytes, generating a gradient of signal that coordinates chondrocyte
differentiation, chondrocyte proliferation and perichondrial development
(Kronenberg, 2003). Previous
work involving chimeras and knockout mice has shown that Ihh regulates
chondrocyte differentiation by stimulating the synthesis of parathyroid
hormone-like peptide (Pthrp; also known as Pthlh - Mouse Genome Informatics)
in the periarticular region (Chung et al.,
1998; St-Jacques et al.,
1999; Vortkamp et al.,
1996). Pthrp, which is also a paracrine signalling molecule,
diffuses back towards the centre of the developing bones and acts on the
parathyroid hormone (Pth)/Pthrp receptor (PPR; also known as Pthr1-Mouse
Genome Informatics) to prevent proliferative columnar chondrocytes
differentiating into postmitotic hypertrophic cells
(Karaplis et al., 1998;
Lanske et al., 1996;
Weir et al., 1996). By doing
this, Pthrp suppresses Ihh expression in early hypertrophic cells and an
Ihh-Pthrp negative feedback loop is established to regulate the distance from
the joint at which chondrocytes leave the proliferative pool and undergo
hypertrophy (Chung et al.,
1998; Vortkamp et al.,
1996). Accordingly, Ihh-depleted mice have abnormal growth plates
with undetectable levels of Pthrp expression in which chondrocytes undergo
hypertrophy closer to the ends of bones and lack the characteristic stacked
columns of proliferating chondrocytes. In addition, Ihh mutants display
Pthrp-independent defects, including dramatic reduction in chondrocyte
proliferation and failure of cortical bone formation
(Razzaque et al., 2005;
St-Jacques et al., 1999).
|
).
It has been shown that vertebrate Hh signalling is mediated through primary
cilia. Cilia are essential for Shh-mediated patterning in early limb bud and
neural tube development (Huangfu et al.,
2003; Liu et al.,
2005). Important components of the pathway, suppressor of fused
(Sufu) and the Gli proteins, localise to primary cilia and Smo translocates to
cilia upon pathway activation (Corbit et
al., 2005; Haycraft et al.,
2005). The recent generation of a Prx1Cre (also known as
Prrx1-cre) conditional allele of Ift88 has enabled study of
cilia-dependent processes in mesenchyme-derived tissues of the limb and
confirms, as anticipated, that cilia are also required for Ihh signalling
(Haycraft et al., 2007). In
mutants with disrupted anterograde or retrograde intraflagellar transport,
Gli3 processing is abnormal with an increase in full-length Gli3 and decreased
levels of Gli3R (Haycraft et al.,
2005; Huangfu and Anderson,
2005; Liu et al.,
2005; May et al.,
2005). Despite the increase in full-length Gli3 in intraflagellar
transport (IFT) mutants, all have low expression levels of the readouts of the
pathway, Gli1 and Ptch1
(Huangfu et al., 2003;
Liu et al., 2005), proving
that inhibition of Gli processing is not sufficient to produce transcriptional
activation and that additional IFT-dependent events are required to promote
Gli activator functioning.
Here we report that Evc localises at the base of primary cilia and demonstrate that defective transduction of Ihh signalling underlies the skeletal phenotype associated with Evc mutation. Our findings show that Evc acts as a positive mediator of Hh signalling downstream of Smo, but that it is not essential for Gli processing.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Antibody production
Amino acids 459-999 of the mouse Evc protein (GenBank CAB76567) were
expressed with a 6xHis tag in E. coli and purified by
Ni2+ chelation chromatography (Novagen). The protein (150 µg)
was used to immunise sheep (Diagnostic Scotland). Total IgGs were prepared
from final serum (Protein G HiTrap, Amersham) and anti-Evc antibodies purified
by affinity to the antigen conjugated to AminoLink Coupling Gel (Pierce).
Immunofluorescent staining
Tissue was frozen in OCT and cryosections (10 µm) dried onto charged
slides. mIMCD3 cells were cultured using standard conditions. Tissue sections
and cells were fixed in 4% (w/v) paraformaldehyde (PFA) in PBS at 4°C.
Primary antibodies were: sheep polyclonal anti-Evc (5 µg/ml); mouse
monoclonal anti-
-tubulin (1:800, GTU-88, Sigma) and anti-acetylated
tubulin (1:2000, 6-11B-1, Sigma). Secondary antibodies were: donkey anti-sheep
Alexa Fluor 594 (Molecular Probes) and goat anti-mouse FITC (Sigma). Samples
were mounted in Vectashield with DAPI (Vector) and images captured on an
Axioplan 2 fluorescence microscope (Zeiss).
Whole-mount X-Gal staining, skeletal preparations and X-ray analysis
Evc +/-, Evc -/- and wild-type control
embryos were stained for ß-gal activity with X-Gal as previously
described (Hogan et al., 1994;
Schatz et al., 2005). E15.5
embryo heads and newborn hindlimbs were stained with X-Gal and embedded in a
gelatine/albumin mix for vibratome sectioning. Sections (100 µm) were
mounted in 50% (v/v) glycerol in PBS for photographing. Skeletal preparations
of P6 mice were conducted as previously described
(Kessel and Gruss, 1991).
Digital radiographs of euthanised P1 and P18 mice were collected at X-ray
exposures of 30 kV for 5 seconds and 45 kV for 10 seconds, respectively. Bones
measurements were compared by ANOVA.
Histology and BrdU analysis
Embryo limbs were fixed in 4% PFA in PBS and paraffin embedded. P16 limbs
were decalcified in 4% formaldehyde:nitric acid:H2O (1:1:8) for a
week prior to embedding. Sections (7 µm) were stained with
Haematoxylin-Eosin or by trichromic staining (0.13% Light Green SF Yellowish,
0.16% Orange G, 0.2% fuchsin acid). Standard von-Kossa method was used for
detection of mineralisation. E17.5 pregnant females were sacrificed 5 hours
after receiving an intraperitoneal injection of BrdU cell proliferation
labelling reagent (GE Healthcare). Proliferating cells were detected on 7
µm paraffin sections by immunocytochemistry (BrdU Staining Kit, Zymed).
BrdU-positive and BrdU-negative nuclei were counted in columnar chondrocytes
of Evc-/- and wild-type mice (12 and seven sections,
respectively). The percentage BrdU-positive cells was calculated for each
section and data compared by ANOVA.
In situ hybridisation
Hindlimbs from E16.5 littermates were fixed overnight in 4% PFA in PBS,
decalcified in 14% (w/v) EDTA in PBS and paraffin embedded. Sections (10
µm) were hybridised using 2x107 cpm/ml
35S-labelled antisense riboprobes, except for Col2a1 and
Col10a1, which were digoxigenin labelled (Roche). Hybridisation steps
were as in Lescher et al. (Lescher et al.,
1998). After stringency washes, dehydrated sections were coated
with nuclear emulsion (K5, Ilford) and exposed for 2-4 weeks at 4°C.
Signal was developed in Kodak D-19 developer and fixer and sections
counterstained with nuclear Fast Red. Hybridisation probes were obtained by
request, except for Gli1 (AB025922) and mouse PPR
(NM_011199), which were RT-PCR amplified. Bright-field images were obtained on
a Nikon DS-5L1 digital camera and dark-fields produced in Photoshop
(Adobe).
Purmorphamine treatment of cells and quantification of target genes
Mouse embryonic fibroblasts (MEFs) were established from
Evc-/- and control E14.5 embryos
(Todaro and Green, 1963).
Chondrocytes were isolated from tibial epiphysial cartilage dissected from two
E17.5 Evc-/- and two Evc+/+
littermates as described (Shingleton et
al., 2000) with the following modifications to enzyme incubation
times: hyaluronidase, 5 minutes; trypsin, 10 minutes; collagenase, 5 hours.
Chondrocytes were incubated in DMEM containing 10% fetal calf serum at
2x106 cells in 6-well plates for a maximum of 7 days
following dissection. For Hh pathway induction experiments,
1x105 cells were grown overnight in 12-well plates and
treated for 72 hours with either 2 µM purmorphamine (10 mM stock in DMSO,
Calbiochem) or DMSO alone. Simultaneous RT-PCR amplification of Ptch1
(nt 1944-2303; NM_008957) and Hprt (nt 108-294; NM_013556) in MEF
cDNA was performed for 22 cycles under standard PCR conditions. Assays were
carried out twice on cultures derived from three Evc-/-,
two Evc+/+ and one Evc+/- embryo.
Ratios of Ptch1 to Hprt band intensity were determined for
each culture before and after treatment and compared by ANOVA. For the
quantitative PCR experiments, chondrocyte cDNA samples were loaded onto Taqman
Low Density Array (TLDA) microfluidic cards (ABI, Forster City, CA). The
following primers and probes were used: Gli1 (ABI assay ID.
Mm00494645_m1), Ptch1 (ABI assay ID. Mm00436026_m1), Evc
(ABI assay ID. Mm00469587_m1), Evc2 (ABI assay ID. Mm00507589_m1) and
ß-actin (Actb; ABI assay ID. Mm00607939_s1). Relative
quantification of genes was performed using the ABI Prism 7900HT Sequence
Detection System and expressed as arbitrary units as determined by 2-(Ct
gene-Ct ß-actin).
Gli3 western blot
Protein lysate from E14.5 limbs was prepared in RIPA buffer supplemented
with one Mini Complete Protease Inhibitor tablet (Roche) and 0.1M PMSF. Total
protein was electrophoresed on 7.5% SDS-PAGE gels and transferred to Hybond-C
membrane (Amersham). Gli3 was detected using rabbit anti-Gli3 (1:200, a gift
of B. Wang, Cornell University, NY) followed by goat anti-rabbit peroxidase
(1:5000, Jackson ImmunoResearch) and SuperSignal West Dura Extended Duration
Substrate (Pierce). Protein loading was assessed using rabbit
anti-ß-catenin (BD Biosciences).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-tubulin and acetylated tubulin as
markers of centrioles and cilia axoneme, respectively, we found that Evc
localises at the distal end of the maternal centriole (the basal body), at the
base of the axoneme (Fig.
2A,B), suggestive of localisation to the transition zone.
Specificity of the Evc immunostaining was demonstrated by absence of signal in
chondrocytes from Evc-/- mice
(Fig. 2C). Cilia were present
in Evc-/- chondrocytes, demonstrating that Evc is not
essential for ciliogenesis (Fig.
2C).
Evc is expressed in the developing skeleton and the orofacial region
During construction of the Evc targeting vector, we inserted a
lacZ reporter cassette directly under control of the Evc
promoter to determine the Evc spatiotemporal expression pattern
during development. By whole-mount X-Gal staining of
Evc+/- mouse embryos, we first observed lacZ
expression at E11.5 in the orofacial region in the lateral nasal process,
maxillary and mandibular processes (Fig.
3A-D), followed at E12.5 by expression in the mesenchymal
condensations of the skeletal system at the time they initiate chondrocyte
differentiation (Fig. 3C). At
E15.5, vibratome sectioning of the orofacial region demonstrated ß-gal
activity in the cartilages of the nasal septum and nasal capsule and those
enclosing the Jacobson organ (Fig.
3E). Strong ß-gal activity was also evident in the upper and
lower lip mesenchyme and in the mesenchyme outlining the growing bones of the
maxilla and mandible (Fig. 3H).
By E15.5, lacZ expression was general to all the cartilaginous
components of the skeleton, including the chondrocranium, the vertebrae, the
rib cage and the axial skeleton (Fig.
3F,G). In the developmentally more advanced elements of the axial
skeleton, lacZ expression was narrowed to the chondrocytes of the
epiphysis and the perichondrium and absent in the central bone metaphyses.
However, in the phalanges in which chondrocytes have not yet undergone
hypertrophy, the entire element was lacZ-positive. In order to
determine which type of chondrocytes express Evc, we studied newborn
skeletal growth plates in which chondrocyte layers are more easily
distinguishable. Vibratome sectioning of stained newborn skeletal growth
plates showed ß-gal activity in the perichondrium and in resting and
proliferating chondrocytes (Fig.
3M,N), but no activity in the prehypertrophic and hypertrophic
chondrocytes. Finally, we observed ß-gal activity in the cranial sutures,
suggesting a role in both endochondral and intramembranous ossification. We
detected lacZ expression in nails
(Fig. 3L), the dermal papilla
of the vibrissae (Fig. 3J) and
in the mesenchyme surrounding the developing tooth buds
(Fig. 3I), in keeping with the
ectodermal phenotype seen in EvC patients.
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2 tests and thus no prenatal loss of
Evc-/- embryos was detected. However, approximately half
of the Evc-/- offspring were missing 2 days after birth.
These neonatal losses are unlikely to be due to cardiovascular malformation
because no overt cardiovascular malformations were observed on histological
examination of 14 Evc-/- mice (eight at E14.5, one at
E15.5, four at E18 and one newborn) (results not shown). Surviving
Evc-/- mutants were unable to feed on a normal diet, but
were able to survive to adulthood when supplied with soft, well-hydrated food;
they did not breed. The phenotype of Evc-/- mice
recapitulates the human disorder with short ribs, short limbs and dental
anomalies, although they do not have polydactyly
(Fig. 4A,B). Preliminary
examination of the teeth revealed variability in the abnormalities of the
incisors, including absence of the upper incisors and a single upper incisor.
We frequently observed a smaller first molar, in comparison with wild-type
dentition (Fig. 4C). We focussed on analysis of the skeletal abnormalities and commenced by studying radiographs and skeleton preparations. At birth, there was no difference between the head-to-tail length of wild-type and mutant mice, but the rib cage was narrow and the radius, ulna, femur and tibia were shorter in the mutant mice (see Table S1 in the supplementary material). At P18, radiographs revealed that the mutant mice are smaller than littermates, that the rib cage is small and that shortening of the radius, ulna and tibia is more pronounced than for the humerus and femur, indicating the same pattern of bone shortening in the mouse mutant as in patients with EvC (Fig. 4A). These observations were confirmed by measurements taken from the radiographs, with decreased radius/humerus (P=0.00065), tibia/femur (P=0.0045) and rib cage/body length (P=0.00853) ratios at P18 (see Table S1 in the supplementary material). Alcian Blue-Alizarin Red skeletal staining also demonstrated this pattern of limb shortening and in addition showed an irregular margin between bone and cartilage in the basiocciput and costochondral junctions (Fig. 4B), as well as premature mineralisation of some of the pedicles between the vertebral bodies and vertebral laminae (Fig. 4B, arrows).
Abnormal growth plate development in the long bones of Evc-/- mice
Histological analysis of embryonic growth plates revealed shorter
proliferative and hypertrophic chondrocyte layers in the epiphyses of the long
bones with hypertrophy of chondrocytes occurring closer to the articular
region than in the wild-type controls. Chondrocytes in the proliferative zone
in Evc-/- mice had the characteristic flattened shape
(Fig. 5A). We consistently
observed an abnormality in the shape of the upper end of the tibia
(Fig. 5A,C), comparable to that
seen in EvC patients. We studied mineralisation by von-Kossa staining and
detected delayed formation of the periosteum adjacent to the prehypertrophic
and hypertrophic chondrocytes (Fig.
5B, red arrows). Similarly, trichromic staining in older mice
demonstrated delayed formation of the secondary ossification centres
(Fig. 5C).
Ihh signalling is diminished in Evc-/- mice, although Gli3 processing appears to be normal
The phenotypic observations of epiphyseal shortening due to chondrocytes
hypertrophying nearer to the articular region, defective perichondrium to
periosteum induction and mineralisation of synchondroses are all factors
compatible with impaired Ihh signalling in mice
(Hilton et al., 2005;
Koziel et al., 2005;
Razzaque et al., 2005;
St-Jacques et al., 1999). In
addition, Evc localises to the base of cilia, structures that mediate Hh
signalling (Huangfu and Anderson,
2005). We therefore decided to determine whether defective Hh
signalling underlies the Evc-/- skeletal phenotype and
assessed expression of Ihh signalling molecules in the growth plates of
Evc-/- mice by radioactive in situ hybridisation. We found
Ihh expression in prehypertrophic chondrocytes of
Evc-/- mice to be normal. However, expression of the Ihh
downstream targets Ptch1 and Gli1 was drastically reduced in
the adjacent perichondrium and proliferating chondrocytes, providing evidence
of defective Ihh signalling (Fig.
6A). To corroborate this, we derived mouse embryonic fibroblasts
(MEFs) from Evc-/- and control littermates and assessed
induction of Ptch1 expression under exogenous stimulation of the
pathway with purmorphamine (Sinha and
Chen, 2006; Wu et al.,
2004). RT-PCR results from MEF cultures showed that the
Ptch1 response of purmorphamine-treated Evc-/-
MEFs was diminished compared with wild-type MEFs
(Fig. 6B). The mean ratio of
Ptch1:Hprt expression was significantly different between
Evc-/- and wild-type fibroblast cultures grown in the
presence of purmorphamine (ANOVA, P=0.003). In addition, we assessed
Ptch1 and Gli1 transcript levels following purmorphamine
treatment by quantitative RT-PCR in chondrocytes derived from
Evc-/- and wild-type littermates. The Ptch1
(Fig. 6C) and Gli1
(Fig. 6D) response of
Evc-/- purmorphamine-treated chondrocytes was greatly
diminished compared with control cells. Since purmorphamine is a Hh agonist
that targets Smo, these studies confirm abnormal Hh signalling in Evc
mutants and demonstrate an intracellular defect downstream of Smo.
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Cilia and IFT proteins have been shown to be required for proteolytic
processing of Gli3. Gli3R is dramatically decreased and full-length Gli3
dramatically increased in IFT mutant embryos at E9.5-11.5
(Haycraft et al., 2005;
Liu et al., 2005). Western
blot analysis of protein extracted from E10.5 Evc-/- and
control embryos revealed normal levels of both full-length Gli3 and Gli3R
(results not shown). However, as there is no detectable ß-gal activity in
Evc+/- or Evc-/- embryos at this point
in development, we tested whether Evc is required for Gli3 processing in limb
extracts from E14.5 embryos. Levels of Gli3, particularly full-length Gli3,
have previously been shown to be very low in the E14.5 limb
(Hilton et al., 2005). We did
not observe a change in the levels of either full-length Gli3 or Gli3R in
E14.5 Evc-/- limb extracts when compared with littermate
controls (Fig. 6E).
Proliferation is normal but differentiation from columnar to hypertrophic chondrocytes occurs prematurely in Evc-/- mice
One of the features of Ihh-knockout mice is a striking
proliferation deficiency known to be caused by the increase in Gli3 repression
(Hilton et al., 2005;
Koziel et al., 2005). To test
whether proliferation is affected in Evc mutants, we undertook in
vivo BrdU labelling experiments followed by immunohistochemistry. No
significant difference was observed in the percentage of BrdU-positive
chondrocytes between Evc-/- and wild-type mice in the
proximal tibia at E17.5 (Fig.
7A). As proliferation and apoptosis (data not shown) do not seem
to be affected, we hypothesised that the growth plate shortening in
Evc-/- mice is likely to be due to premature chondrocyte
differentiation.
|
), we went on
to study Pthrp signalling and found that Pthrp expression in
articular chondrocytes, which is normally maintained by Ihh signalling, was
decreased (Fig. 7B). We
observed normal expression of the Pth/Pthrp receptor, PPR, in the
prehypertrophic zone, but expression in the osteoprogenitor cells of the
perichondrium (Naski et al.,
1998) (Fig. 7B,
arrows) was reduced in the Evc-/- growth plate
(Fig. 7B), in keeping with the
mineralisation defect in the perichondrium evidenced by the von-Kossa
staining.
To further clarify whether Evc-/- growth plate
shortening is due to premature differentiation from distal to columnar
chondrocytes, or from columnar to hypertrophic chondrocytes, or both, we
supplemented the histological analysis by studying Fgfr1 expression
as a marker of distal and hypertrophic chondrocytes and Fgfr3
expression as a marker of columnar and early hypertrophic cells. The
hybridisation results showed that there was no shortening of the distal
Fgfr1 expression domain in Evc-/- mice, but
marked shortening of the zone expressing Fgfr3
(Fig. 7C), from which we
conclude that differentiation from distal to columnar chondrocyte, which is
influenced by Gli3R (Koziel et al.,
2005), is proceeding normally and hypertrophic differentiation is
occurring prematurely.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Koziel et al., 2005). The
proliferation deficiency in the growth plate of Ihh-/-
mice is fully rescued in the double knockouts and is therefore attributable to
increased Gli3 repression. Gli3R levels are normal in
Evc-/- mice and, consistent with this, chondrocyte
proliferation in Evc-/- mice is normal. By contrast, in
the growth plates of the Ihh-/-,Gli3-/- mice,
there is no Gli1 expression and there is only partial recovery of the
reduced levels of Pthrp and Ptch1 expression. Similarly, the
bone collar defect is not completely rescued. Lack of function of Gli
activators is the likely cause for these features of
Ihh-/- mice, which are not restored in the double
knockouts (Hilton et al.,
2005; Koziel et al.,
2005). In Evc-/- mice, there is low expression
of Gli1, Ptch1 and Pthrp in the growth plate as observed in
the double knockouts. Likewise, both Evc-/- mice and the
double knockouts have defective bone collar formation as manifest by decreased
PPR expression in the perichondrium and in the von-Kossa staining. We
postulate that Evc-/- mice are likely to be deficient in
the same Ihh-dependent Gli activator functions that are absent in the
Ihh-/-,Gli3-/- mice.
|
). The
extended range of signalling has been attributed to the absence of
Ptch-mediated ligand sequestration (Chen
and Struhl, 1996; Lewis et
al., 2001). We observed similar expression of Ptch1 and
Gli1 in the weakly Evc-expressing cells surrounding the
articular surface in Evc-/- mice
(Fig. 6A). This indicates that
in mice lacking Evc, Hh signalling is most markedly perturbed in those cells
that would normally have the highest levels of Evc expression.
Bone shortening in Evc-/- as a result of premature chondrocyte hypertrophy
Growth plate, and hence bone, shortening could result from one or more of
the following: decreased proliferation, increased apoptosis, premature
differentiation either of distal to columnar chondrocyte or from proliferating
to hypertrophic chondrocytes. In Evc-/- mice,
proliferation appears normal, consistent with the normal Gli3R levels observed
in western blotting, and apoptosis also appears normal. Differentiation from
distal to columnar chondrocyte, which is regulated by Gli3R levels
(Koziel et al., 2005), is also
normal in Evc-/- mice. As shown by the considerable
reduction in size of the Fgfr3 expression domain, the
Evc-/- growth plate shortening is explained by the shorter
region of proliferating columns of chondrocytes, which in turn is due to the
premature onset of hypertrophic differentiation. Hypertrophic differentiation
is regulated by Pthrp, itself a target of Gli activation
(Koziel et al., 2005). Thus,
we can conclude that premature hypertrophic differentiation in
Evc-/- chondrocytes is due to decreased Pthrp
expression secondary to defective Hh signalling.
Comparison of Evc-/- with IFT mutants
Defects in IFT cause embryonic lethality and it is only with the advent of
mice carrying a conditional Ift88 allele and the Prx1Cre
transgene that it has been possible to study endochondral bone formation in
IFT mutants (Haycraft et al.,
2007). Evc-/- and the Prx1Cre Ift88
conditional mice both have decreased activation of Ihh targets Ptch1
and Gli1 in the growth plates of long bones and delayed bone collar
formation, features that are not fully rescued by Gli3R derepression in the
Ihh-/-,Gli3-/- mice and that are expected to be
associated with defective Gli activation. Evc mutants differ from the
IFT conditionals with respect to Gli3R, which is decreased in the IFT nulls.
Chondrogenic clumps of cells surrounding the perichondrium are described in
Prx1Cre Ift88 conditional mice, a feature that we have not observed
in the Evc mutant and not reported in the
Ihh-/-,Gli3-/- mice. An additional difference
between IFT and Evc mutants relates to Ihh expression, which
appears normal in Evc-/- mice but is reduced in the
Prx1Cre conditional allele of Ift88.
Evc involvement in Shh signalling
Evc-depleted mice phenocopy most of the EvC features and therefore
represent a good model for this syndrome. They reproduce the skeletal and
dental anomalies of the condition and the perinatal lethality. Similar
neonatal losses in Ihh-/- and Pthrp-/-
mice have been attributed to respiratory failure secondary to the small rib
cage (Karaplis et al., 1998;
St-Jacques et al., 1999). The
lack of polydactyly in Evc-/- mice came as a surprise as
EvC individuals invariably have postaxial polydactyly of the hands. Neither
Gli1 nor Gli2 mutant mice manifest limb patterning defects
(Park et al., 2000). However,
the spontaneous and targeted Gli3 mutants have polydactyly, and
polydactyly also occurs in human disorders caused by Gli3 mutations
such Pallister-Hall syndrome and Greig cephalopolysyndactyly syndrome
(Ehlen et al., 2006). In many
of the mutants with polydactyly, including the IFT mutants, both Gli3
activator function and Gli3 repression are perturbed. It is apparent that the
balance between Gli3 activation and Gli3 repression across the limb bud is
crucial in specifying the number of digits
(Ahn and Joyner, 2004;
Wang et al., 2007). The fact
that we did not detect lacZ expression in the limb buds of
Evc-/- mice when patterning is being established indicates
that Evc expression in the limb buds is low compared with that in
bone anlagen, and this might explain the absence of polydactyly in
Evc-/- mice. Regardless, as anteroposterior patterning in
the limb bud is regulated by Shh rather than Ihh, the polydactyly seen in EvC
individuals suggests that EVC may also play a crucial role in
transduction of Shh signalling during human limb bud morphogenesis. The
occasional observation of a single central incisor in
Evc-/- mice is also suggestive of defective Shh signalling
in the mouse given that this is a feature seen in association with
holoprosencephaly due to SHH mutations. Similarly, temperospatial
expression of lacZ in tissues where Shh is expressed, such as the
orofacial region and vibrissae (Bitgood and
McMahon, 1995), is consistent with Evc participation in
the transduction of the Shh signal.
Conclusion
We have shown that Evc localises at the distal end of the basal body and
the base of the axoneme and that it is integral to Ihh signalling in
developing bones. Intracellular transduction of Hh signal is not yet fully
understood. There have been surprises - knockdowns of two of the components of
the pathway first identified in Drosophila, Sufu and Fu, indicate
that their roles differ between Drosophila and vertebrates
(Svard et al., 2006;
Varjosalo et al., 2006). Evc
adds to the differences between Drosophila and vertebrates, as there
are no recognisable Evc homologues in organisms other than vertebrates. In the
last few years, new players have been identified acting downstream of Smo in
mice [Rab23 (Eggenschwiler et al.,
2006) and tectonic (Reiter and
Skarnes, 2006)], in zebrafish [Iguana (also known as Dzip1 - ZFIN)
(Sekimizu et al., 2004;
Wolff et al., 2004)] and in
chick [Talpid (Davey et al.,
2006)], the precise role of which remain to be elucidated. Here we
show that Evc is a novel basal body component of Hh signalling indispensable
for normal endochondral growth, acting downstream of Smo to facilitate
transcription of the Ihh-regulated genes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/16/2903/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Instituto de Investigaciones Biomédicas, Consejo
Superior de Investigaciones Científicas, Arturo Duperier 4, 28029
Madrid, Spain
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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s organ; ul, upper lip; oc, oral
cavitity; ui, upper incisor; mx, maxilla; ma, mandible; mc, Meckel's
cartilage; tb, temporal bone; li, lower incisor; dp, dermal papillae. Growth
plate chondrocytes labelling: R, resting; P, proliferative; PH,
prehypertrophic; H, hypertrophic; B, bone.
