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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001586
1 Department of Orthopaedic Surgery, Thomas Jefferson University College of
Medicine, Philadelphia, PA 19107, USA.
2 Department of Developmental Biology, Harvard School of Dental Medicine,
Boston, MA 02138, USA.
3 Department of Cell Biology, University of Alabama at Birmingham, Birmingham,
AL 35294, USA.
Authors for correspondence (e-mails:
eiki.koyama{at}jefferson.edu;
maurizio.pacifici{at}jefferson.edu)
Accepted 30 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Key words: Kif3a, Primary cilia, Cranial base synchondroses, Hedgehog signaling, Syndecans, Growth plate, Intramembranous ossification, Ectopic cartilage, Exostoses, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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). These organelles, which were first described
decades ago, are microtubule-containing non-motile structures protruding from
the surface of most vertebrate cells
(Scherft and Daems, 1967;
Sorokin, 1968). Recent studies
have shown that primary cilia are important regulators of cell and tissue
function, and of developmental processes. Mutations in the structural and
functional components of primary cilia can cause serious human conditions,
including polycystic kidney disease and retinal degeneration
(Ansley et al., 2003;
Lin et al., 2003;
Pazour et al., 2000).
Similarly, altered primary cilia function can have damaging repercussions on
limb patterning, neural tube formation and other major developmental processes
(Huangfu et al., 2003;
Murcia et al., 2000;
Zhang et al., 2003). The
importance and centrality of primary cilia in both health and disease reflect
the fact that these organelles and their IFT system have turned out to mediate
and transduce intercellular chemical, physical and biological signals
(Davenport and Yoder, 2005),
including those elicited by hedgehog proteins
(Corbit et al., 2005;
Haycraft et al., 2005;
May et al., 2005). These
proteins are well-known regulators of multiple developmental processes
(Bitgood and McMahon, 1995;
McMahon et al., 2003). They
are lipid-containing factors, secreted by donor cells, that diffuse through
their surroundings and exert action on short- and long-range target cells
(Gritli-Linde et al., 2001;
Yin et al., 2002). The
proteins act by binding to the cell surface receptor Patched 1 (Ptch1), and
signals are transmitted by the receptor smoothened via the zinc-finger
transcription factors Gli1, Gli2 and Gli3. Current thinking, which has been
reviewed recently (Huangfu and Anderson,
2006; Singla and Reiter,
2006), is that, following hedgehog interaction with Patched 1,
smoothened is activated and translocated to the primary cilia via the IFT
system, resulting in the production of Gli activator forms that would
translocate to the nucleus and activate hedgehog gene target expression. In
the absence of hedgehog proteins, smoothened would remain sequestered and
inactive, and Gli proteins would be processed into repressor forms and move to
the nucleus to inhibit hedgehog target gene expression.
A well-documented target of hedgehog signaling is the developing axial and
limb skeleton (Bitgood and McMahon,
1995; Koyama et al.,
1996; Lanske et al.,
1996). As a recent study from our groups shows, hedgehog signaling
is also very important for the early formation, growth and function of the
cranial base and its synchondroses (Young
et al., 2006). The synchondroses are cartilaginous palindromic
structures each consisting of two mirror-image growth plates and termed
ethmoidal, intrasphenoidal, spheno-occipital or intraoccipital according to
their anatomical location. They are important for cranial base development and
overall growth patterns (Ingervall and
Thilander, 1972; Roberts and
Blackwood, 1983), and defects in synchondrosis functioning are
likely to contribute to craniofacial deformities such as those seen in Crouzon
and Apert syndromes (Chen et al.,
1999; Kreiborg et al.,
1993). We found that synchondrosis development is severely
affected in mice lacking the Indian hedgehog (Ihh) gene.
Ihh-/- cartilaginous cranial bases were short and
deficient in extracellular proteoglycan matrix. The mutant synchondroses
displayed abnormal chondrocyte proliferation, hypertrophy and topographical
arrangement with respect to the perichondrium; as a consequence, endochondral
ossification was deranged. In several but not all respects, these
abnormalities are reminiscent of those seen in developing
Ihh-/- long bones
(St-Jacques et al., 1999),
pointing to the pivotal nature and general requirement of hedgehog action in
skeletogenesis. Given their roles in hedgehog signaling, primary cilia should
be as essential for skeletal development and growth as hedgehog proteins, and
previous genetic studies certainly support that premise
(Murcia et al., 2000;
Olsen et al., 2005;
Xiao et al., 2006;
Zhang et al., 2003). In
particular, limb and cranial skeletal abnormalities were found in conditional
mouse mutants deficient in Kif3a or in the cilia-associated
mechanosensory protein polycystin-1 (Pkd1), as well as in mice bearing a
missense mutation in the Pkd1 gene
(Olsen et al., 2005;
Xiao et al., 2006). A more
extensive recent study indicated that long bone anlagen in mouse embryo limbs
deficient in Ift88 or Kif3a
(prx1cre;Ift88fl/n or
prx1cre;Kif3afl/n) are short, exhibit accelerated
chondrocyte hypertrophy and lack an intramembranous bone collar
(Haycraft et al., 2007). In
the present study, we analyzed mice deficient in Kif3a in cartilage
and found that cranial base development and growth were also impaired,
particularly at postnatal stages. However, mutant synchondroses displayed a
delay in chondrocyte hypertrophy, excessive intramembranous ossification along
their perichondrial border and ectopic cartilage masses. These defects were
associated with a topographical re-distribution of hedgehog signaling that was
quite feeble within the mutant growth plates themselves, but was prominent and
widespread all along the perichondrial border, thus most likely responsible
for the excessive ossification and ectopic cartilage formation. When we
examined mice deficient in Ihh in cartilage at similar postnatal
stages, their synchondrosis defects minimally resembled those of
Kif3a-deficient mice, pointing to unique contributions and regulatory
roles of primary cilia in synchondrosis development and function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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) with
Col2a1-Cre transgenic mice
(Ovdhinnikov et al., 2000).
The genotype of transgenic mice was determined by PCR analyses of genomic DNA
isolated from tail or liver. The Col2a1-Cre mice were identified by
using Primer1: 5'-TGCTCTGTCCGTTTGCCG-3' and Primer2:
5'-ACTGTGTCCAGACCAGGC-3', generating one band of approximately 720
bp. The loxP alleles or Kif3a were identified using three primers:
Primer1: 5'-TCTGTGAGTTTGTGACCAGCC-3'; Primer2:
5'-AGGGCAGACGGAAGGGTGG-3'; and Primer3:
5'-TGGCAGGTCAATGGACGCAG-3'. Homozygous Kif3a-deficient
mice were born with Mendelian frequency and were studied at postnatal days
(P)0, P7 and P15.
Homozygous mice deficient in Ihh in postnatal cartilage were
produced by sequentially mating Ihhfl/fl mice
(Maeda et al., 2007) with
Col2a1-CreER transgenic mice
(Nakamura et al., 2006). The
genotyping of mice was performed by PCR using the following specific primers:
fl-Ihh forward: 5'-AGCACCTTTTTTCTCGACTGCCTG-3';
fl-Ihh reverse: 5'-TGTTAGGCCGAGAGGGATTTCGTG-3'; Cre
275: 5'-CGCGGTCTGGCAGTAAAAACTATC-3'; Cre 603:
5'-CCCACCGTCAGTACGTGAGATATC-3'. After an initial denaturation for
8 minutes at 94°C, amplification cycles consisted of denaturation at
94°C for 30 seconds, annealing at 68°C for 30 seconds and 45 seconds
extension at 72°C for 35 cycles, followed by a final extension for 10
minutes at 72°C. The expected amplicons for the wild-type Ihh
allele were 320 bp, 400 bp for the floxed Ihh allele and 328 bp for
the Cre allele. To verify Cre activity and specificity
postnatally, Col2a1-CreER mice were mated with Rosa R26R reporter
mice and resulting double-transgenic mice were injected with
4-hydroxy-tamoxifen (Sigma) at P0 and evaluated for lacZ activity at
P1, P4, P7 and P14 by histochemistry
(Nakamura et al., 2006;
Maeda et al., 2007).
Micro-computed tomography, histology and immunohistochemistry
Skulls were fixed in buffered 4% paraformaldehyde overnight at 4°C,
rinsed and subjected to micro-computed tomography (µCT) using a µCT40
SCANCO Medical system. Samples were scanned using a 36 mm holder at 45 kV of
energy, 12 µm scanning thickness and medium resolution. Two-dimensional
slice images were selected and used to generate three-dimensional
reconstructions with the following parameters: filter width sigma=0.8, support
level=1.0 and threshold=173. The same values were used to analyze wild-type
and mutant samples at each specified time point. Three-dimensional images were
rotated at specific angles to generate lateral and bird's-eye views of the
cranial base.
For histology and immunohistochemistry, fixed skulls were rinsed in DEPC
(diethyl pyrocarbonate)-treated water and decalcified in 0.1 M Tris, pH 7.5
buffer containing 0.1% DEPC, 10% EDTA-4 Na, and 7.5% polyvinyl pyrolidione
(PVP). For routine histological analysis, 5 µm sections were stained with
hematoxylin and Eosin or Fast green using standard procedures. To visualize
primary cilia by immunofluorescence staining, 10 µm paraffin sections were
incubated with a 1:200 dilution of anti-acetylated 
-tubulin monoclonal
antibody (Sigma, Clone 6-11B-1) followed by biotinylated secondary antibodies
and Cy3-conjugated streptavidin (Vector Laboratories) to yield a signal.
Sections were counterstained with YOPRO-3 iodide (612/631) or
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen) to
reveal nuclei. To visualize hedgehog protein distribution, paraffin sections
were subjected to antigen retrieval by incubation in 0.05% citraconic
anhydride pH 7.4 at 93°C for a total of 30 minutes
(Namimatsu et al., 2005).
Following blocking with 5% normal goat serum, sections were incubated with a
1:500 dilution of rabbit anti-mouse Shh (Ab80) in 5% normal goat serum at
4°C overnight; this antibody mixture had been pre-spun at 10,000
g at 4°C for 10 minutes prior to use. After overnight
incubation, sections were rinsed in phosphate-buffered saline (PBS) and
incubated with biotinylated goat anti-rabbit IgGs followed by Cy3-Streptavidin
according to the manufacturer's recommendation. Nuclear counterstaining was
carried out with diluted DAPI solution for 5 minutes at room temperature.
Gene expression analysis
Serial paraffin tissue sections were pretreated with 10 µg/ml proteinase
K (Sigma) for 10 minutes at room temperature, post-fixed in 4%
paraformaldehyde, washed with PBS containing 2 mg/ml glycine and treated with
0.25% acetic anhydride in triethanolamine buffer
(Koyama et al., 1996).
Sections were hybridized with antisense or sense 35S-labeled probes
[approximately 1x106 disintegrations per second
(DPM)/section] at 50°C for 16 hours. Mouse cDNA clones included: histone
H4C [nucleotides (nt.) 549-799; AY158963]; Pthrp (nt. 66-1386;
NM_008970); osteopontin (Spp1; nt. 1-267; AF515708); osterix
(Sp7; nt. 40-1727; NM_130458); Vegf (Vegfa; nt.
115-539; gi/249858); Ihh (nt. 897-1954; MN_010544); Patched 1
(Ptch1; nt. 81-841; NM_008957); smoothened (nt. 450-1000; BC048091);
collagen X (Col10a1; nt. 1302-1816; NM009925); and collagen II
(Col2a1; nt. 1095-1344; X57982). After hybridization, slides were
washed with 2x sodium chloride-sodium citrate buffer (SSC) containing
50% formamide at 50°C, treated with 20 µg/ml RNase A for 30 minutes at
37°C and washed three times with 0.1x SSC at 50°C for 10
minutes/wash. Sections were dehydrated with 70, 90 and 100% ethanol for 5
minutes/step, coated with Kodak NTB-3 emulsion diluted 1:1 with water, and
exposed for 10-14 days. Slides were developed with Kodak D-19 at 20°C and
stained with hematoxylin. Dark- and bright-field images were captured using a
digital camera, and dark-field images were pseudo-colored using Adobe
Photoshop software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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-tubulin confirmed that
primary cilia were obvious in control chondrocytes
(Fig. 1F, arrowheads) but rare
in Kif3a-deficient chondrocytes
(Fig. 1M, arrowhead), attesting
to the effectiveness of the action of Col2a1-Cre. In addition,
primary cilia were equally evident in perichondrial tissue of both control and
Kif3a-deficient cranial bases
(Fig. 1G,N, arrowheads),
attesting to the specificity of Col2a1-Cre action being clearly
limited to cartilage.
Gene expression analysis reveals dysfunction of mutant growth plates
Upon closer microscopic inspection, control synchondroses, such as the
spheno-occipital synchondrosis (Fig.
2A), displayed characteristic growth plate zones of small, round,
resting chondrocytes (Fig. 2B,
rz); flat-shaped proliferating chondrocytes
(Fig. 2B, pz); and
oval pre-hypertrophic chondrocytes and large mature hypertrophic chondrocytes
(Fig. 2C, phz and
hz). The hypertrophic zone was followed by primary bone spongiosa
intermixing with marrow (Fig.
2D, arrowheads). By sharp contrast, Kif3a-deficient
growth plates lacked well-defined zones
(Fig. 2I). In particular,
small-sized resting chondrocytes and flat-shaped chondrocytes were
conspicuously absent; instead, a mixed population of slightly enlarged and
oval-to-round chondrocytes was present
(Fig. 2J,K). In addition, the
hypertrophic zone was very small (Fig.
2K) and there was an abrupt transition from cartilage to marrow,
and a concurrent reduction in primary spongiosa and endochondral bone
(Fig. 2L)
(Xiao et al., 2006). In line
with the absence of flat-shaped cells, mutant growth plates contained few
proliferating chondrocytes, as indicated by gene expression of the mitotic
marker histone 4C (Fig. 2M-O),
whereas the cells were abundant and organized in two distinct proliferative
zones in control growth plates (Fig.
2E-G, arrowheads). Indeed, gene expression of Pthrp, a
major regulator of growth plate chondrocyte proliferation
(Lanske et al., 1996;
Vortkamp et al., 1996), was
strong in controls (Fig. 2H),
but was low in mutant growth plates (Fig.
2P).
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;
Zelzer et al., 2002).
Deficiency in primary spongiosa (Fig.
4F) was reflected in the fact that expression of such bone markers
and regulators as osteopontin (also known as Spp1 - Mouse Genome
Informatics), collagen I (Col1a1), Runx2 and osterix (also
known as Sp7 - Mouse Genome Informatics) was quite low in mutant
(Fig. 4G-J) versus control
(Fig. 4B-E) specimens. Despite
all of these serious changes, mutant chondrocytes still exhibited strong
collagen II (Col2a1) gene expression, as did controls
(Fig. 3A,F), signifying that
the basic differentiated phenotype in mutants was largely preserved.
Excessive intramembranous ossification and ectopic cartilage formation
As they develop and mature in the growth plate, pre-hypertrophic and
hypertrophic chondrocytes induce the formation of intramembranous bone in
adjacent perichondrial tissues, a process that is attributable to Ihh
expression (Koyama et al.,
1996; Nakamura et al.,
1997; St-Jacques et al.,
1999). Control synchondroses, such as the spheno-occipital
synchondrosis, conformed to this general rule. It displayed
fast-green-positive intramembranous bone flanking the Ihh-rich
pre-hypertrophic and hypertrophic zones
(Fig. 5A, arrowhead), and which
expressed collagen I, Runx2 and osterix
(Fig. 5B-D, arrowheads);
importantly, there was no appreciable bone flanking the proliferative and
resting zones, as was to be expected (Fig.
5A-D, arrows). By contrast, intramembranous bone had formed all
along the growth plate in companion Kif3a-deficient synchondrosis,
thus spanning the entire flank of the synchondrosis and bridging the
neighboring occipital and sphenoidal bones
(Fig. 5E-H). This excess
intramembranous bone undoubtedly corresponds to the regions of mineralized
tissue initially detected by µCT (Fig.
1I, arrows) and appreciable also by general histology
(Fig. 1K,L, double
arrowheads).
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). Ectopic
cartilage masses were never seen in tissues flanking control synchondroses,
and their chondro-perichondrial boundary was extremely well-defined and
un-violated (Fig. 6A-C).
Aberrant topography of hedgehog signaling
What could have triggered excessive intramembranous ossification and
ectopic cartilage formation near mutant growth plates? An obvious possibility
was that hedgehog signaling, and in particular the topography of signaling,
had changed in mutant specimens, triggering broader responses in neighboring
perichondrial tissues. To test this possibility, we determined the expression
patterns of Ihh and of its target genes Patched 1 and Gli1
in control and Kif3a-deficient synchondroses. We chose to focus on
P0, hoping to detect changes in signaling patterns as early as possible. In
controls, the three genes displayed predictable and distinct expression
patterns, with abundant Ihh transcripts in pre-hypertrophic zones
(Fig. 7A,B,G,H, phz)
and Patched 1 and Gli1 transcripts in preceding proliferative zones
(Fig. 7C,D,I,J, pz),
reflecting the well-established and direct role of Ihh in chondrocyte
proliferation (Shimo et al.,
2004; St-Jacques et al.,
1999). There was also clear Patched 1 and Gli1 expression
in perichondrial tissue flanking the pre-hypertrophic and hypertrophic zones
where the intramembranous bone collar normally forms
(Fig. 7C,D,I,J, single
arrowhead), but perichondrium flanking the resting and proliferative zones was
negative (Fig. 7C,D,I,J, double
arrowhead). Smoothened transcripts were present throughout the growth plates
(Fig. 7E,K). In mutant
synchondroses, Ihh expression was still appreciable at this stage
(Fig. 7M,N,S,T). However,
expression of both Patched 1 and Gli1 was markedly decreased and the
transcripts were scattered within the growth plates
(Fig. 7O,P,U,V), signifying
that hedgehog signaling was feeble and diffuse. On the other hand, the Patched
1 and Gli1 transcripts were actually quite evident along the entire
length of perichondrial tissues (Fig.
7O,P,U,V, arrowhead), thus closely mirroring the location of
excessive intramembranous bone formation. Smoothened expression was largely
unaffected (Fig. 7Q,W).
Immunohistochemistry showed that Ihh distribution in the mutants had also been
re-arranged. In controls, the protein was abundant in pre-hypertrophic
chondrocytes, as well as in neighboring proliferative chondrocytes and in the
inner perichondrial layer (Fig.
8A,B, arrowhead), and was undetectable in the upper growth plate
region and flanking perichondrium (Fig.
8B, double arrowhead) as expected
(Yin et al., 2002). In the
mutants, Ihh formed a much more expansive gradient that extended throughout
the upper growth plate region and along the entire length of perichondrium
(Fig. 8C,D, double arrowhead).
Specificity of staining was reaffirmed by the fact that sections from
Ihh-null mice were negative (Fig.
8E,F).
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;
The et al., 1999), and on
syndecan 3 in particular, which we found to participate in such roles in long
bone growth plates (Shimazu et al.,
1996; Shimo et al.,
2004). In controls, the syndecan 3 gene was expressed in the
proliferative zones of synchondrosis growth plates
(Fig. 7F,L) along with Patched
1 and Gli1, in line with the idea that this syndecan may aid and
delimit Ihh action on chondrocyte proliferation
(Pacifici et al., 2005).
However, there was barely detectable syndecan 3 gene expression in
Kif3a-deficient synchondroses
(Fig. 7R,X).
Skeletal aberrations are caused by postnatal Ihh deficiency
The severe cranial base and synchondrosis abnormalities triggered by
Kif3a deficiency in cartilage raised the issue of whether they had a
unique character or whether they would resemble those elicited by a deficiency
in related genes, such as Ihh. Accordingly, we created conditional
postnatal mice lacking Ihh in cartilage by treating neonatal
Ihhfl/fl;Col2a-CreER mice with tamoxifen. Mice were
sacrificed at P7 and P15 (hereafter termed Ihh-deficient mice);
neonatal Ihhfl/fl littermates were treated with tamoxifen
in parallel and served as controls. Ihh-deficient skulls exhibited
anteroposterior shortening and abnormal synchondroses
(Fig. 9E) relative to controls
(Fig. 9A), reminiscent of the
defects seen in Kif3a-deficient skulls, described above. At variance
with the latter, however, the Ihh-deficient growth plates were
entirely occupied by collagen X-expressing hypertrophic chondrocytes
(Fig. 9F) and there was no
detectable intramembranous ossification in the flanking perichondrial tissues
(Fig. 9G, arrowhead) relative
to controls (Fig. 9B,C).
Defects were exacerbated by P15, at which point much of the
Ihh-deficient synchondroses had been replaced by endochondral bone
and marrow, and remnants of growth plate tissue were confined to a lateral and
inconspicuous position (Fig.
9H), phenomena not seen in P15 Kif3a-deficient
specimens.
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).
Kif3a deficiency affects, as severely, the behavior and functional
organization of synchondrosis growth plates, resulting in a quasi-stasis of
the chondrocyte maturation process and retardation of endochondral bone
formation. Concurrently, there were unexpected and unpredicted alterations in
developmental events occurring in synchondrosis perichondrial tissues,
including excessive intramembranous ossification - most likely impacting very
negatively on cranial base growth. Only some of these defects resembled those
caused by a conditional deficiency of Ihh, indicating that Kif3a and
primary cilia make unique contributions to the normal progression of cranial
base and synchondrosis development, organization and function.
Growth plates are complex multi-facet structures in which chondrocyte
shape, orientation and polarity, chondrocyte proliferation and phenotype, and
chondrocyte maturation and hypertrophy must all be regulated in a coordinated
manner. A well-established mechanism delineated by work on developing long
bones is the PTHrP-Ihh axis, in which PTHrP produced by periarticular cells
under the influence of Ihh regulates the rate of chondrocyte proliferation
(Lanske et al., 1996). In line
with this model, Ihh gene ablation in mouse embryos causes the
downregulation of Pthrp expression and reduced proliferation
(St-Jacques et al., 1999).
This mechanism can certainly account for the decrease in chondrocyte
proliferation in Kif3a-deficient growth plates, which express low if
any PTHrP. This would imply that synchondroses conform to, and obey, the
PTHrP-Ihh axis (Young et al.,
2006) and that a normal complement of Kif3a and primary cilia is
needed to maintain this axis. Reduced proliferation was accompanied by the
absence of flat-shaped chondrocytes, which normally constitute the
proliferative zone; by the absence of distinct resting, pre-hypertrophic and
hypertrophic zones; and by the absence of a columnar cell organization. Such
nearly chaotic organization of Kif3a-deficient growth plates thus
suggests that the mutant chondrocytes may lack key topographical, navigational
or mechanical mechanisms and devices that enable them to perceive location or
cues within the growth plate and adopt appropriate zone-specific polarity,
shape and phenotype. One such mechanism could be the planar cell polarity
pathway (PCP), a form of non-canonical Wnt pathway mediated by primary cilia
that is essential for cell orientation and mitotic spindle alignment in organs
such as the inner ear, kidney tubules and cranial cartilage
(Dabdoub and Kelley, 2005;
Fischer et al., 2006;
Park et al., 2006). The
importance and centrality of the PCP pathway in several systems make it likely
that it operates in growth plates as well
(Olsen et al., 2005), where it
could orient the chondrocyte mitotic spindle and division plane, which are
usually perpendicular to the longitudinal axis of the growth plate
(Dodds, 1930), or where it
could promote the related process of columnar organization. Notably, because
both the PCP pathway and hedgehog proteins act via primary cilia, chondrocytes
could control their own polarity and orientation in relation to, and in
correlation with, their responses to Ihh, which is distributed in the form of
gradients within the growth plate
(Gritli-Linde et al., 2001;
Koziel et al., 2004;
Yin et al., 2002).
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) or in the postnatal Ihh-deficient
synchondroses described here in which there is exuberant and widespread
chondrocyte hypertrophy followed by endochondral bone. One possibility is that
the Kif3a-deficient chondrocytes are unable to respond to factors
that regulate their maturation and hypertrophy, including
ß-catenin-dependent Wnt proteins
(Enomoto-Iwamoto et al., 2002;
Mak et al., 2006;
Tamamura et al., 2005), the
signaling and action of which may involve primary cilia
(Germino, 2005).
Alternatively, the topographical disorganization discussed above could have
led to general intermingling and intermixing of chondrocytes with different
phenotypic properties and maturation status within the mutant growth plates,
creating conditions in which the cells interfere with the phenotype of each
other. Previously, we showed that, when immature chondrocytes are co-cultured
with hypertrophic chondrocytes, the hypertrophic phenotype is suppressed
(D'Angelo and Pacifici, 1997).
It is important to note and discuss here the findings recently described by
Haycraft et al. (Haycraft et al.,
2007). These authors suggested that chondrocyte hypertrophy was
accelerated in the long bone anlagen of prx1cre;Ift88 or
prx1cre;Kif3a mouse embryo limbs, based on histological measurements
of growth plate zones. These findings would appear to be in sharp contrast to
the severe delay in chondrocyte hypertrophy that we observe in our
col2cre;Kif3a synchondroses, but there are several plausible
explanations for these apparent differences. One is that the developmental
consequences of a broad deletion of primary cilia function by prx1cre
may differ from those elicited by the more targeted cartilage-specific
approach we use here. A second intriguing possibility is that the overall
roles of primary cilia in developing long bones may somewhat differ from those
in the cranial base. Third, Haycraft et al. did not use the several markers of
chondrocyte maturation and hypertrophy and endochondral ossification that we
use in our study, and effects on chondrocyte maturation and hypertrophy may
have been underestimated. Lastly, some differences could reflect the fact that
prenatal stages were examined in that study, whereas we focused on neonatal
and early postnatal stages.
One of the most intriguing and unexpected defects seen in
Kif3a-deficient synchondroses is the formation of excessive
intramembranous bone and ectopic cartilage masses, which are both readily
appreciable by P7. These defects are preceded by alteration in
hedgehog-signaling topography, which is delineated by Patched 1 and
Gli1 expression along the entire length of perichondrial tissues.
Given that hedgehog proteins can stimulate osteogenesis and chondrogenesis
(Enomoto-Iwamoto et al., 2000;
Nakamura et al., 1997), excess
hedgehog signaling in perichondrial tissues could have directly caused
excessive intramembranous bone and cartilage formation. What remains unclear,
however, is why there is excess signaling all along the perichondrium to begin
with, particularly starting at P0 when Ihh expression is still
confined to fairly restricted growth plate zones (see
Fig. 7). One possibility is
that the excess signaling is an autonomous local response of the perichondrium
to phenotypic defects in the adjacent growth plates. Alternatively, it could
be due to the mutant chondrocytes themselves. For example,
Kif3a-deficient chondrocytes may lack means to control the location
and distribution of Ihh, allowing it to diffuse broadly, form a more extensive
gradient, reach the entire length of the perichondrium, and trigger excessive
cartilage and bone formation. This scenario and chain of events (summarized in
our model in Fig. 10) are
sustained by our observation that Kif3a-deficient growth plates
express little, if any, (a) Patched 1, which sequesters and restricts hedgehog
signaling to target fields (Jeong and
McMahon, 2004) or (b) syndecan 3, a component of heparan sulfate
proteoglycan-based mechanisms limiting and restricting hedgehog diffusion and
action in growth plates or other structures
(Callejo et al., 2005;
Koziel et al., 2004;
Shimo et al., 2004).
In this regard, it is interesting to point out a recent study involving
mice lacking one allele of Ext2, a Golgi-associated
glycosyltransferase needed for heparan sulfate synthesis
(Stickens et al., 2005).
Ext2 heterozygous mice were found to be deficient in heparan sulfate
and exhibited ectopic cartilaginous masses near their growth plates, thus
mimicking exostoses present in hereditary multiple exostosis (HME) patients,
who often have EXT2 or EXT1 mutations and reduced heparan
sulfate levels (Hecht et al.,
1995). The exact link between heparan sulfate deficiency and
exostosis formation is still poorly understood. In the Ext2 study
above, it was proposed that heparan sulfate deficiency reduces fibroblast
growth factor (FGF) signaling and tilts the balance in favor of BMP signaling,
resulting in chondrocyte misbehavior, weakening of the bone collar and
formation of exostosis masses (Stickens et
al., 2005). Our data, showing reduced syndecan 3 expression in the
growth plate and excess hedgehog signaling and protein in flanking
perichondrial tissues, point to hedgehog proteins and defective Kif3a-related
mechanisms as culprits in exostosis formation as well. A similar role for
hedgehog protein in exostosis formation was proposed in a study of mouse
embryos bearing an Ext1 hypomorphic mutation
(Koziel et al., 2004). We do
not know, however, how Kif3a deficiency and lack of primary cilia
lead to a reduction in syndecan 3 expression. In previous studies, we did find
that syndecan 3 overexpression in vivo and in vitro causes excess and
widespread Ihh expression, suggesting that the two genes are
coordinated and that such coordination may have been disrupted by
Kif3a deficiency (Pacifici et
al., 2005; Shimo et al.,
2004). It is also possible that the reduction in syndecan 3
expression is independent of Kif3a deficiency, but both of these
effects would contribute to the phenotype. Whatever the explanation, it will
be interesting to clarify whether Kif3a deficiency also affects the
expression of syndecan 4 and syndecan 2, which are normally expressed in the
proliferative zone and perichondrial tissues, respectively
(David et al., 1993;
Pacifici et al., 2005), and/or
expression of other heparan sulfate proteoglycans, including glypicans and
perlecan. Given that hedgehog proteins, bone morphogenetic protein (BMPs),
FGFs and Wnts are all heparan sulfate-binding factors
(Bernfield et al., 1999), the
data may provide insights into how the various heparan sulfate proteoglycans,
with their distinct binding specificity and expression patterns
(Esko and Selleck, 2002), may
help to mediate and coordinate the functions of the many signaling factors and
pathways that converge to orchestrate the progression of skeletal development
and how defects in these circuits could cause pathologies, including HME.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Orthopaedic Surgery, University of
California, Los Angeles, CA, USA
|
REFERENCES |
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