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First published online June 6, 2008
doi: 10.1242/10.1242/dev.019950
Research Report |
1 Division of Genetics, Genomics and Development, Department of Molecular and
Cell Biology, Center for Integrative Genomics, University of California,
Berkeley, CA 94720, USA.
2 Departments of Internal Medicine (Rheumatology) and Biochemistry, Rush
University Medical Center, 1725 Harrison Street, Chicago, IL 60612, USA.
* Authors for correspondence (e-mail: sohaskey{at}berkeley.edu; harland{at}berkeley.edu)
Accepted 27 April 2008
SUMMARY
Properly positioned synovial joints are crucial to coordinated skeletal movement. Despite their importance for skeletal development and function, the molecular mechanisms that underlie joint positioning are not well understood. We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired chondroitin sulfation and abnormal metabolism of the chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton.
Key words: Chondrogenesis, Synovial joints, Interzone, Gdf5, Chondroitin sulfate, Extracellular matrix, Mouse embryo
INTRODUCTION
Synovial joints segment the developing cartilage template into individual
skeletal elements while providing flexibility and structural stability between
these elements. Histologically, the earliest sign of synovial joint formation
is the appearance of the interzone
(Mitrovic, 1977
), which occurs
at approximately embryonic day (E) 12.5-13.5 in mouse digits. Several
signaling pathways are implicated in the molecular specification of the joint
interzone, including those downstream of the secreted proteins growth
differentiation factor 5 (Gdf5) and Wnt9a
(Hartmann and Tabin, 2001;
Pacifici et al., 2005;
Khan et al., 2007). By
contrast, the molecular mechanisms that control spatial positioning of the
joint interzone are not well understood. Here we identify a previously
uncharacterized protein, which we have named JAWS (joints abnormal with
splitting), as a novel and essential coordinator of cartilage formation and
synovial joint positioning.
MATERIALS AND METHODS
Animals
The KST245 mouse embryonic stem cell line, containing an insertion of the
pGT1TMpfs gene trap vector in the Jaws/Impad1 locus, was isolated and
characterized as described (Mitchell et
al., 2001). Jaws F1 heterozygotes were backcrossed to
C57BL/6 mice for six generations before intercrossing. Genotyping was
performed by X-gal staining of yolk sacs and/or tail biopsies, or by RT-PCR
using primers flanking the insertion site (sequences available upon
request).
Expression studies
Jaws transcripts were detected by northern blotting of
Trizol-extracted total RNA, probed with a cDNA fragment spanning nucleotides
726-1158 of the Jaws mRNA (GenBank Accession Number BC145952).
Alternatively, Jaws expression was assessed by quantitative RT-PCR
using SYBR Green reagents (Applied Biosystems). For immunoblotting of E13.5
embryo lysates (60 µg), a polyclonal antiserum was raised against the
peptide epitope N-CRESNVLHEKSKGKTREGADD-C corresponding to residues 83-102 of
the JAWS protein.
Histology, in situ hybridization and immunohistochemistry
Alcian Blue and Alizarin Red staining of skeletal preparations and
histological sections, X-gal staining of embryos, and in situ hybridization
were performed as described (Nagy,
2003). To enhance contrast against the Hoechst counterstain, the
radioactive ISH images were photographed in bright-field and then
pseudo-colored red using the `Colorize' option within the `Hue/Saturation'
command in Adobe Photoshop 7.0. Immunohistochemistry on paraffin sections was
carried out after antigen retrieval, using Alexa Fluor-conjugated secondary
antibodies and the following primary antibodies: mouse aggrecan 12/21/1-C-6
and link protein 9/30/8-A-4 (Developmental Studies Hybridoma Bank), mouse
β-catenin (BD Biosciences), mouse chondroitin sulfate CS-56
(Sigma-Aldrich), rabbit collagen II (Abcam) and rat CD44S (Chemicon).
Apoptotic cells were labeled using the In Situ Cell Death Detection kit
(Roche).
Fluorophore-assisted carbohydrate electrophoresis (FACE)
The composition and amount of chondroitin sulfate and heparan sulfate were
analyzed as described (Plaas et al.,
2001; Gao et al.,
2004). Values for total CS, HS and HA were normalized to wet
tissue weight. FACE data were analyzed by a two-tailed Student's
t-test, and values were considered statistically significant at
P<0.05.
Protein extraction and aggrecan immunoblotting
Successive nondissociative extractions (0.15 M NaCl, then 0.15 M NaCl +
0.5% CHAPS) of E14.5 limb tissue were followed by one dissociative extraction
(4 M guanidinium HCl + 0.5% CHAPS) to solubilize matrix-associated proteins.
Loadings for SDS-PAGE were normalized to wet tissue weight, and immunoblots
were processed with polyclonal CDAGWL antibodies directed against the
N-terminal G1 domain of aggrecan. Blots were exposed to film simultaneously
and developed identically.
RESULTS AND DISCUSSION
JAWS is required for the development of the endochondral skeleton
Jaws (also known as Impad1-Mouse Genome Informatics) was
identified in an insertional mutagenesis screen for genes encoding secreted
and transmembrane proteins essential for mammalian development
(Mitchell et al., 2001). The
Jaws/Impad1 locus encodes a predicted protein of 
39 kDa with an
N-terminal hydrophobic domain and a consensus inositol monophosphatase
catalytic motif. Insertion of the gene-trap vector into the third of four
introns fuses the first 213 amino acids of the JAWS protein in-frame with a
βgeo reporter, resulting in less than 0.2% expression of wild-type (WT)
transcripts and negligible expression of the endogenous protein (see Fig. S1
in the supplementary material). Together with the observation that βgeo
fusion proteins generated by this `secretory trap' accumulate in cytoplasmic
inclusion bodies (Skarnes et al.,
1995; Mitchell et al.,
2001), these data suggest that the Jaws insertion creates
a null or strongly hypomorphic allele.
|
Mice carrying one copy of the Jaws insertional mutation
(Jaws+/-)appeared phenotypically indistinguishable from
wild-type littermates. By contrast, mice homozygous for the insertion
(Jaws-/-) died perinatally with cleft secondary palate
(see Fig. S2 in the supplementary material). Jaws-/-
neonates exhibited severe dwarfism with stunted limbs, hypoplastic ribcages
and rounder shortened craniofacies (Fig.
1B,C). The cranial vault and clavicles, which form without a
cartilage intermediate, were largely unaffected
(Fig. 1C; data not shown).
These results reveal that the skeletal defects in Jaws-/-
embryos were restricted to elements formed through endochondral ossification.
Defects in chondrogenesis were apparent by E12.5 in
Jaws-/- embryos (see Fig. S3 in the supplementary
material), despite robust expression of the chondrogenic markers Sox9,
Sox5 and Col2a1 (see Fig. S4 in the supplementary material)
(Akiyama et al., 2002). This
latter observation suggests that JAWS is dispensable for the formation of
prechondrogenic condensations from limb bud mesenchyme.
In the embryonic growth plate, chondrocytes align in a pseudocolumnar
arrangement along the longitudinal axis according to their differentiation
status, which is reflected in the demarcation of four histologically and
molecularly distinct chondrocytic zones: resting, proliferative,
prehypertrophic and hypertrophic
(Kronenberg, 2003).
Histologically, Jaws-/- chondrocytes lacked the
pseudocolumnar organization of their wild-type counterparts
(Fig. 1D). Mutant growth plates
were hypocellular, with discontinuities in Alcian Blue staining revealing
regions devoid of extracellular matrix (ECM). After E15.5, ectopic
chondrocytes were regularly seen impinging on the primary ossification center
in Jaws-/- embryos
(Fig. 1D, arrow).
JAWS deficiency delays chondrocyte maturation and disrupts long-range Ihh signaling in the growth plate
To analyze growth plate maturation in molecular detail, we examined markers
of chondrocyte differentiation by in situ hybridization at E14.5
(Fig. 2A). Expression of the
proliferating chondrocyte marker Col2a1 was largely excluded from the
central Col10a1-expressing hypertrophic zone in wild-type long bones,
whereas in Jaws-/- embryos Col2a1 expression
partially overlapped this Col10a1 domain. During hypertrophic
differentiation, maturing chondrocytes abruptly switch from Col2a1 to
Col10a1 expression; thus, this overlap in the two domains suggests
that many Jaws-/- chondrocytes were in a delayed
intermediate state of already expressing Col10a1 without yet having
downregulated Col2a1 expression
(St-Jacques et al., 1999).
Similarly, wild-type expression of Ihh or Pthr appeared in
symmetrical domains of prehypertrophic chondrocytes flanking the central
Col10a1 domain (Kronenberg,
2003); however, one continuous domain of expression overlapping
that of Col10a1 was seen for each gene in Jaws-/-
embryos. Altered expression of two downstream targets of Ihh signaling,
Ptch1 and Pthrp, was also evident. Ptch1 expression
was unchanged in the perichondrium and proliferating chondrocytes proximal to
the Ihh source in Jaws-/- embryos; however, expression of
Ptch1 in resting and periarticular chondrocytes was markedly
diminished, as was expression of Pthrp
(Fig. 2A, arrows). This finding
implies normal short-range, but disrupted long-range, Ihh signaling in the
Jaws-/- growth plate, probably resulting from compromised
ECM integrity (see below). Collectively, these data indicate a delay in
chondrocyte maturation in Jaws-/- embryos. Terminal
hypertrophic chondrocyte differentiation was also delayed in E15.5
Jaws-/- cartilage, as evidenced by a persistent central
domain of Col10a1 expression and reduced expression of the terminal
differentiation markers Mmp13 and osteopontin (Spp1)
(Fig. 2B). Delayed chondrocyte
maturation was most conspicuous in the hindlimb, where the knee joint failed
to form and where the tibia retained a homogeneous population of small,
Col2a1-expressing chondrocytes at E18.5
(Fig. 3A; see Fig. S5 in the
supplementary material). Finally, expression of the osteoprogenitor markers
Runx2 (Kronenberg,
2003) and Tcf1 (Glass,
2nd et al., 2005) was similar in wild-type and
Jaws-/- long bones, excluding the tibia
(Fig. 2A; data not shown),
suggesting that subsequent osteogenesis, when it occurred, initiated
normally.
|
E13.5, extended from the presumptive
metacarpophalangeal/metatarsophalangeal joint to the distalmost phalanx in all
digits, appearing discontinuous in forelimbs while bisecting and spanning each
hindlimb element. Two main possibilities could explain the occurrence of these
cavities: either they result from aberrant cell death, or they represent
ectopic joints. To distinguish these possibilities, we first examined the
early joint marker Gdf5 by whole-mount in situ hybridization and
found that Gdf5 expression extended longitudinally throughout these
cavities (Fig. 3B; Fig. S6 in
the supplementary material). Likewise, expression of all joint markers
examined, including Gli3, Wnt9a, Sulf1, Cutl1 (Cux1),
Stc1, Hip1 and activated β-catenin
(Guo et al., 2004;
Hill et al., 2005;
Pacifici et al., 2005;
Mak et al., 2006;
Zhao et al., 2006;
Khan et al., 2007) overlapped
the Gdf5 expression domain (Fig.
3C and data not shown), suggesting that cells within these
cavities exhibited a joint-like fate. Histologically, these cells resembled
wild-type joint progenitors in having a flattened, more mesenchymal appearance
than surrounding chondrocytes (Fig.
3C; data not shown). TUNEL analysis demonstrated similar numbers
of apoptotic cells in wild-type and Jaws-/- joints at all
stages examined (E12.5-E17.5; see Fig. S7 in the supplementary material, and
data not shown), further excluding cell death as a disproportionate
contributor to the joint phenotype. Likewise, consistent cartilage staining in
the digits of E12.5 Jaws-/- autopods prior to the
appearance of the joint interzone suggested that ectopic joints were not a
secondary consequence of diminished ECM formation (see Fig. S3 in the
supplementary material; data not shown). Moreover, no changes in the
expression of limb polarity markers were evident earlier in E11.5 or E12.5
Jaws-/- embryos (see Fig. S8 in the supplementary
material).
If the longitudinal cavities in Jaws-/- digits
represent joints, then they should not only express joint markers but also
exclude markers of neighboring cell types. Indeed, expression of the
chondrogenic markers collagen 2, aggrecan and link protein was excluded from
prospective Jaws-/- joints
(Fig. 3D and data not shown)
(Schwartz and Domowicz, 2002).
Similarly, expression of Bmpr1b, which is normally restricted to
articular surfaces flanking the wild-type joint interzone at E14.5
(Baur et al., 2000;
Yi et al., 2000), was seen in
an analogous pattern bordering the longitudinal domain of Gdf5
expression in Jaws-/- digits
(Fig. 3D). Thus, joint-like
structures in Jaws-/- digits do not express chondrogenic
markers but do maintain their immediate proximity to
Bmpr1b-expressing articular chondrocytes. In addition, these
joint-like structures did not express markers of hypertrophic chondrocytes
(Col10a1) or osteoblasts (Runx2), while expressing wild-type
levels of the tendon and ligament marker Scx
(Fig. 3E)
(Schweitzer et al., 2001).
Collectively, these molecular and histological data establish the longitudinal
cavities in Jaws-/- digits as ectopic joints. The
persistence of ectopic joints in neonatal Jaws-/- mice
defines JAWS as a crucial novel regulator of synovial joint orientation and
positioning. Because misorientation of the interzone necessarily causes an
ectopic joint, we use the terms `orientation' and `positioning'
interchangeably here.
|
).
By contrast, CD44 immunostaining was largely absent from ectopic
Jaws-/- joints, despite its presence in more proximal
joints (Fig. 3F; data not
shown). This result indicates a requirement for JAWS in the timely progression
to cavitation, leading to a functional synovial joint.
Impaired chondroitin sulfation and aberrant aggrecan metabolism in Jaws-/- cartilage
From a mechanistic perspective, several features of the
Jaws-/- phenotype are consistent with defects in
chondroitin sulfation pathways. In particular, misaligned chondrocytes,
hypocellularity and ECM discontinuities characterize the growth plates of
animals lacking chondroitin-4-sulfotransferase 1 (C4st1;
Chst11-Mouse Genome Informatics) or the chondroitin sulfate (CS)- and
heparan sulfate (HS)-rich proteoglycan perlecan
(Klüppel et al., 2005;
Schwartz and Domowicz, 2002).
Moreover, mutant mice lacking components of the CS synthesis or transport
machinery are chondrodysplastic (Schwartz
and Domowicz, 2002). For these reasons, we examined whether CS was
spatially or quantitatively altered in Jaws-/- embryos.
Immunostaining for CS demonstrated highest levels in the perichondrium and
articular zones of E14.5 limbs (Fig.
4A; data not shown), with intense uniform extracellular staining
in wid-type digits. By contrast, a faint punctate pattern was observed in
Jaws-/- digits, much of which appeared to localize
intracellularly (Fig. 4A,
insets).
We then used fluorophore-assisted carbohydrate electrophoresis to analyze
and quantify glycosaminoglycan content in E14.5 limbs. Consistent with our
immunostaining data, significant decreases in the proportions of both
chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) were evident in
Jaws-/- limbs, with concomitant increases in unsulfated
chondroitin (C0S) (Fig. 4B).
Importantly, no change in heparan sulfation was observed
(Fig. 4C). Moreover, quantities
of total CS (including C0S), HS and HA normalized to wet tissue weight were
comparable in wild-type and Jaws-/- limbs
(Fig. 4D). The expression of
C4st1, Slc26a2 and Papss2-key regulators of CS biosynthesis
and/or transport-was also unchanged, implying that JAWS does not influence CS
levels through transcriptional regulation of these genes (see Fig. S9 in the
supplementary material). As described above, this specific diminution of
chondroitin sulfation is consistent with the Jaws-/-
growth plate defects and suggests a molecular mechanism by which JAWS
coordinates chondrogenesis and joint positioning. As extracellular HS is
important for shaping the Ihh morphogen gradient
(Koziel et al., 2004), these
data also argue that the disruption of long-range Ihh signaling in the
Jaws-/- growth plate
(Fig. 2A) does not result from
a change in HS levels but from a generalized loss of ECM integrity.
|
).
The CS chains of aggrecan are essential for its efficient secretion and for
binding and proteolytic cleavage by the aggrecanase Adamts4
(Kiani et al., 2001;
Tortorella et al., 2000).
Therefore, we assessed whether Jaws-/- embryos show
impaired expression and/or turnover of aggrecan. Aggrecan immunostaining
revealed mottled, variable expression throughout E14.5
Jaws-/- cartilage, in contrast to the robust consistent
wild-type staining pattern (Fig.
4E and data not shown). Notably, mRNA and protein levels of two
other abundant cartilage ECM proteins, collagen 2 and link protein, were
unchanged, suggesting that abnormal aggrecan expression was not a secondary
consequence of delayed chondrocyte maturation (see Fig. S10 in the
supplementary material; data not shown). To confirm and extend this aggrecan
result, an antibody (anti-CDAGWL) that recognizes intact aggrecan and several
well-characterized cleavage products was used to analyze limb protein extracts
(Arner, 2002). Under
dissociative conditions, markedly reduced amounts of both full-length aggrecan
(Fig. 4F, arrow) and
C-terminally cleaved aggrecan fragments
(Fig. 4F, bracket) were
extracted from Jaws-/- relative to wild-type limbs. This
reduction was greater in hindlimbs, where more severe chondrogenic and joint
defects were noted (Fig. 3A;
Fig. S5 in the supplementary material). As an internal control, successive
nondissociative extractions yielded similar levels of a 65 kDa aggrecan
fragment that is likely to result from transient expression and rapid cleavage
in extracartilaginous tissue (Fig.
4F, arrowhead) (Arner,
2002). Collectively, these data suggest that impaired CS sulfation
and abnormal aggrecan metabolism contribute to the chondrodysplasia and
aberrant joint positioning in Jaws-/- embryos.
Although the precise mechanism by which JAWS influences CS sulfation and
ECM integrity is unclear, CS sulfation has been reported to modulate cell
adhesion and migration (Zou et al.,
2004), consistent with the misalignment of both growth plate
chondrocytes and the axis of joint formation in Jaws-/-
embryos. Moreover, the particular importance of CS sulfation for
chondrogenesis may explain why mutation of Jaws, a widely expressed
gene, produces defects that are relatively specific to endochondral
ossification.
Importantly, this genetic loss-of-function approach distinguishes our
findings from previous demonstrations of ectopic joint formation caused by
overexpressing Wnt9a or activated β-catenin in vivo or by blocking
5β1 integrin function in limb explant cultures
(Hartmann and Tabin, 2001;
Guo et al., 2004;
Garciadiego-Cazares et al.,
2004). Our findings also contrast with data obtained by
inactivation of the hypoxia-inducible factor 1 (Hif1a) gene
in mouse limb mesenchyme. Conditional deletion of Hif1a using
Prx1-Cre produces longitudinal cavities that are superficially
similar to those seen in Jaws-/- digits; however, unlike
ectopic Jaws-/- joints, Hif1a-null cavities form
prior to the wild-type joint interzone and do not express joint markers such
as Wnt9a and Bmp2
(Amarilio et al., 2007;
Provot et al., 2007).
Gdf5 is expressed primarily in diffuse transverse (rather than
longitudinal) stripes in Hif1a-null digits, leading Provot and
colleagues to conclude that after an initial developmental delay, synovial
joints of normal appearance do form in the digits of these mice. Together,
data presented by both groups argue that the Hif1a-null digit
phenotype reflects delayed joint specification caused by aberrant
differentiation of hypoxic chondrocytes.
By demonstrating the persistence of ectopic joints in Jaws-/- digits, our studies identify JAWS as a novel regulator in the establishment of the interphalangeal joint axes. Because cells in the ectopic Jaws-/- joint express appropriate early interzone markers (Fig. 3B,C), we conclude that the molecular specification and the spatial positioning of the synovial joint interzone are genetically separable processes. Similarly, the restriction of ectopic joints to digits and the lack of knee joints in Jaws-/- embryos support the notion that distinct Jaws-dependent mechanisms govern synovial joint development at different sites in the embryonic skeleton. Overall, Jaws-/- mice provide a unique animal model for exploring the tightly coordinated processes of chondrogenesis and joint morphogenesis, and for better understanding the etiology of joint degeneration leading to debilitating diseases such as osteoarthritis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2215/DC1
ACKNOWLEDGMENTS
We thank Jen-Yi Lee, David Stafford, Danielle Behonick and Pete Savage for critical reading of the manuscript; Edivinia Pangilinan for mouse husbandry; Lisa Brunet for technical expertise and the Cutl1 in situ hybridization probe; and Céline Colnot for instruction on in situ hybridization and the Mmp13 and osteopontin probes. We thank B. de Crombrugghe, M. Hilton and F. Long, S. Dymecki, A. McMahon, Y. Yang, G. Karsenty, P.-T. Chuang, G. DiMattia, R. Johnson, D. Kingsley, G. Martin and M. Scott for in situ hybridization probes. Monoclonal antibodies 12/21/1-C-6 and 9/30/8-A-4 developed by B. Caterson were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by a grant from the NIH (R.M.H.). M.L.S. was a Michael Geisman Fellow of the Osteogenesis Imperfecta Foundation, and a Foundation for Advanced Cancer Studies and Merck Fellow of the Life Sciences Research Foundation.
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UA-galNAc; C4S, 
P<0.0001,
Jaws-/- versus wild type percentage (n=5 pairs
per genotype). (D) Comparable amounts (mean and s.d.) of total
glycosaminoglycans in wild type and Jaws-/- hindlimbs,
normalized to wet tissue weight. Similar results were obtained for forelimbs.
n.d., not determined; HA, hyaluronan. (E) Aggrecan immunostaining in
the proximal end of the humerus (E14.5). (F) Anti-CDAGWL immunoblots of
E14.5 limb protein extracts, showing less full-length aggrecan (arrow,