1 Department of Molecular and Cellular Biology, Harvard University, Cambridge,
MA 02138, USA
2 Endocrine Unit, Massachusetts General Hospital, Harvard Medical School,
Boston, MA 02114, USA
3 Department of Bone and Cartilage Regenerative Medicine, University of Tokyo
School of Medicine, Tokyo, Japan
Author for correspondence (e-mail:
amcmahon{at}mcb.harvard.edu)
Accepted 2 December 2003
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SUMMARY |
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Key words: Osteoblast, Endochondral skeleton, Chondrocyte, Ihh, Smo
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Introduction |
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Indian hedgehog (Ihh), one of the three mammalian homologues of the
Drosophila Hedgehog (Hh) protein, has emerged as a key regulator of
the developing endochondral skeleton. Ihh is primarily expressed by
pre- and early hypertrophic chondrocytes, and it signals to both immature
chondrocytes and overlying perichondrial cells
(St-Jacques et al., 1999;
Vortkamp et al., 1996
).
Studies in both chicken and mouse have established that Ihh controls the onset
of chondrocyte hypertrophy primarily via parathyroid hormone-related protein
(PTHrP) (Karp et al., 2000
;
Lanske et al., 1996
;
St-Jacques et al., 1999
;
Vortkamp et al., 1996
). The
Ihh knockout studies also revealed that Ihh is a potent positive
regulator of chondrocyte proliferation
(St-Jacques et al., 1999
). As
with all Hh proteins, Ihh signaling in the receiving cells requires the
seven-pass transmembrane protein encoded by smoothened (Smo). Recent
genetic manipulations of Smo by both loss-of-function and
gain-of-function approaches have demonstrated that Ihh directly regulates
chondrocyte proliferation (Long et al.,
2001
).
Ihh is also indispensable for osteoblast differentiation in the
endochondral skeleton. This is evidenced by the lack of a bone collar in the
long bones of Ihh null (Ihhn/n) mice
(St-Jacques et al., 1999).
Furthermore, bone collar forms adjacent to ectopic hypertrophic chondrocytes
in an Ihh-dependent fashion in growth plates containing both wild-type and
PTH/PTHrP receptor (also known as Pthr1) null chondrocytes
(Chung et al., 1998
;
Chung et al., 2001
). However,
it is not clear whether the regulation of osteoblast formation by Ihh
indicates a direct requirement for Ihh signaling in osteoblast
differentiation, or occurs merely as a secondary consequence of Ihh signaling
in chondrocytes. In addition, since the long bones in
Ihhn/n embryos fail to develop a marrow cavity
when they die at birth, it has not been possible to determine whether Ihh
signaling is required for the formation of the primary spongiosa.
The bone morphogenetic protein (BMP) family of proteins has long been
implicated in osteogenesis. BMPs are capable of inducing bone de novo when
implanted ectopically in vivo (Wozney et
al., 1988); several BMPs are expressed by chondrocytes and the
perichondrium during normal development
(Pathi et al., 1999
). A direct
role of BMPs in osteoblast differentiation however remains to be established,
as bone induction by ectopic BMPs appears to be mediated by a cartilage
intermediate, recapitulating endochondral bone formation. Moreover, genetic
removal of BMP signaling has not be informative in this regard because of
complications such as early lethality or potential functional redundancy among
family members (Hogan,
1996
).
We have studied the requirement for Ihh signaling in osteoblast development in the long bones. The data suggest that Ihh plays a direct role in promoting osteogenesis, most probably in conjunction with BMPs.
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Materials and methods |
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Morphological analysis
Whole-mount staining of skeletons were performed according to the method of
McLeod with modifications (McLeod,
1980). For chimeric analyses, embryos were sacrificed at various
ages, dissected, and fixed in 4% paraformaldehyde/PBS at 4°C for 4 hours.
For detection of ß-galactosidase activity, tissues were stained with
X-Gal (5-bromo-4-chloro-3-indolyl ß-D-galactoside) as described
previously (Rossert et al.,
1995
). Subsequently, stained tissues were processed, embedded in
paraffin wax, and sectioned at 6 µm. Sections were stained with Hematoxylin
and Eosin (H&E) or Nuclear Fast Red for histological analyses. For
detection of mineralization, sections were stained with 1% silver nitrate (von
Kossa method) and counterstained with Methyl Green, Nuclear Fast Red, or
H&E. To detect cartilage matrix, sections were stained with Safranin-O.
For histological analyses of postnatal limbs, after fixation in 10%
formalin/PBS at room temperature overnight, the samples were decalcified in
20% EDTA at room temperature for 3-4 days with changes of the solution daily.
Decalcified limbs were subsequently processed and sectioned in paraffin wax at
6 µm.
In situ hybridization
Tissues were fixed overnight either in 4% paraformaldehyde/PBS at 4°C
or in 10% formalin/PBS at room temperature, processed, embedded in paraffin
wax, and sectioned at 6 µm. As for histological analyses, postnatal limbs
were decalcified in EDTA (see above) prior to processing and sectioning. In
situ hybridization was performed as described previously
(Lee et al., 1995) by using
complementary 35S-labeled riboprobes.
Cell culture and northern blot analysis
MLB13MYC clone 17 (Rosen et al.,
1994) was obtained from Genetics Institute (Cambridge, MA) and was
grown in DMEM with 10% fetal bovine serum and penicillin/streptomycin at
37°C. To perform an osteoblast differentiation assay, one day after
plating, cells were treated with recombinant Bmp2, 500 ng/ml, or the
N-terminal fragment of Shh, 500 ng/ml, in the presence or absence of
Hh-signaling inhibitors for 1 or 2 days. Recombinant Bmp2 was obtained from
Genetics Institute (Cambridge, MA), and the recombinant N-terminal fragment of
Shh and the recombinant N-terminal fragment of Ihh were obtained from Curis
(Cambridge, MA). Anti-Shh antibody 5E1
(Roelink et al., 1995
)
(Developmental Studies Hybridoma Bank, University of Iowa) was used at 40
µg/ml. Recombinant Noggin (Regeneron Pharmaceuticals, NY) was used at 1
µg/ml. Total cellular RNA was isolated using Trizol reagent (Gibco-BRL,
Gaithersburg, MD). For northern analysis, 30 µg of total RNA was run on a
1.2% gel, blotted onto a nylon membrane, and hybridized with
32P-labeled cDNA probes for osteocalcin, Cbfa1 (gift from
G. Karsenty, Baylor College of Medicine, Houston, TX), Ptc1 (R.
Johnson, Stanford University, Stanford, CA), Ihh
(Echelard et al., 1993
) and
Id1 (R. Benezra, Memorial Sloan-Kettering, New York City, NY).
Northern analysis for each probe was performed three times.
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Results |
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The bone collar defects observed in Cre3; Smon/c and Cre10; Smon/c embryos prompted us to examine whether Cre is expressed in cells of the osteoblast lineage in these mice. In assays using the Rosa26 reporter strain, both Cre3 (data not shown) and Cre10 (Fig. 1K-M) lines elicit evidence of strong Cre activity not only in chondrocytes but also in the perichondrium, the periosteum and in osteoblasts in the bone collar and primary spongiosa (arrowheads in Fig. 1L and M, respectively). Therefore, it is probable that Cre activity in some cells of the osteoblast lineage leads to removal of Smo and results in the bone collar defects.
In order to assess directly whether Ihh signaling in the perichondrium is
indeed compromised in Cre3; Smon/c and Cre10;
Smon/c embryos, we examined expression of patched 1
(Ptc1; also known as Ptch) and Gli1, both known
transcriptional targets of the hedgehog pathway
(McMahon et al., 2003), by in
situ hybridization. In the wild-type embryo at E14.5, both Ptc1 and
Gli1 are highly expressed in the perichondrium abutting
Ihh-expressing chondrocytes, with Gli1 expressed in a
broader domain (Fig. 2A,D). In
Cre3; Smon/c (data not shown) and Cre10;
Smon/c (Fig.
2C,F) embryos however, neither Ptc1 nor Gli1 is
expressed in a normal perichondrial domain, which is identifiable by a darker
counterstaining resulting from a more compact organization of perichondrial
cells (denoted by arrows in Fig.
2C,F). Interestingly, both genes are expressed at high levels in
cells outside this normal domain of perichondrium, indicating robust Ihh
signaling at this new position. Therefore, in Cre3;
Smon/c and Cre10; Smon/c
embryos, removal of Smo from the inner perichondrium renders cells
outside the normal Ihh signaling domain responsive to Ihh. The
extended range most probably reflects the absence of Ptc1-mediated ligand
sequestration in the normal inner perichondrium, as Ptc1 restricts the
movement of Hh proteins (Chen and Struhl,
1996
; Lewis et al.,
2001
). The Cre15; Smon/c embryos, in
contrast, express Ptc1 and Gli1 in a normal perichondrial
domain, albeit at a higher level than wild-type embryos, most probably
reflecting increased levels of Ihh caused by diminished expression of
Ptc1 in chondrocytes (Fig.
2B,E). These results therefore correlate displacement of the
Ihh-responsive domain in the perichondrium with the observed displacement of
the bone collar with respect to the cartilage in Cre3;
Smon/c and Cre10; Smon/c
embryos.
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We next attempted to determine in the Cre10; Smon/c embryos whether the `outer periphery' cells beyond the normal perichondrium manifest the entire program of gene expression of perichondrial cells, since these cells express Cbfa1 in response to Ihh and evidently give rise to the bone collar. Bone morphogenetic protein 3 (Bmp3) is normally expressed in the perichondrium at E14.5 in a highly specific manner, and therefore serves as a useful marker for the perichondrium (Fig. 2I). Whereas Bmp3 expression is abolished in the perichondrium where Smo expression is abrogated, Bmp3 expression is not induced in the `outer periphery cells' (Fig. 2J). Therefore, these cells appear to be distinct from the bona fide perichondrium and moreover, osteogenesis in a bone collar can be induced without Bmp3 expression.
To further corroborate the direct role for Ihh signaling in osteoblast differentiation, we analyzed chimeric embryos derived from Smon/n and wild-type ES cells. Whereas mutant cells (stained blue in Fig. 3A,B) can contribute to the perichondrium of long bones at E17.5, they fail to differentiate into osteoblasts. Rather, Smon/n cells in the perichondrium assume the morphology of chondrocytes (Fig. 3C), deposit cartilaginous matrix (Fig. 3D) and express chondrocytic markers such as collagens type II and X (Fig. 3E,F). Adjacent wild-type perichondrial cells undergo normal osteoblast differentiation even when the neighboring chondrocytes are entirely of the Smon/n origin (Fig. 3B). As expected, the Smon/+ cells contribute normally to the bone collar of chimeric embryos (Fig. 3J, arrowhead). These results suggest a direct cell-autonomous requirement for Smo activity to promote osteoblast development and prevent chondrocyte formation in a perichondrially located precursor cell.
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Direct requirement for Hh signaling in the formation of primary spongiosa
We next examined development of the primary spongiosa in
Col1(II)-Cre; Smon/c animals. In
the wild-type pups at postnatal day 3 (P3), the primary spongiosa forms a
network of irregular pink-stained (Hematoxylin and Eosin) bone spicules that
extend from the terminal hypertrophic chondrocytes into the bone marrow
(Fig. 4A,C). Before reaching
the marrow cells that are characterized by darkly stained nuclei and little
cytoplasm, the bone spicules are interspersed with cells of lightly stained
nuclei and basophilic cytoplasm, presumably cells of the osteoblast lineage
(note region between the green lines, Fig.
4C). In contrast, the Cre3; Smon/c
(data not shown) and Cre10; Smon/c
(Fig. 4B,D) pups at P3
completely lack the matrix of the primary spongiosa as well as the non-marrow
cells normally adjacent to the matrix (note region between the green lines,
Fig. 4D). Consequently,
hypertrophic chondrocytes directly abut the marrow cells. A similar defect in
the primary spongiosa was observed at E18.5 with the Cre3;
Smon/c and Cre10; Smon/c
embryos (data not shown). In contrast, the Cre15;
Smon/c embryos develop a normal primary spongiosa
(data not shown).
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We next assessed whether the defects in osteoblast differentiation in the
primary spongiosa correlate with disruption of Hh signaling. At P3, the growth
region cartilage of the tibia in Cre10; Smon/c
animals is considerably shorter than in the wild type, because of a decrease
in chondrocyte proliferation (Long et al.,
2001). However, Ihh is characteristically expressed by
pre- and early hypertrophic chondrocytes in the mutant
(Fig. 5B) as in wild-type
littermates (Fig. 5A). In
wild-type pups, Pct1 is expressed strongly at the chondro-osseous
junction by a subpopulation of the cells interspersed among the bone spicules
(Fig. 5C,E). In the Cre10;
Smon/c littermates however, no Ptc1
expression is detected in the similar region abutting the terminal
hypertrophic chondrocytes (Fig.
5D), consistent with the loss of these cells. Therefore, Hh
signaling in cells at the chondro-osseous junction correlates with normal
development of the primary spongiosa.
|
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In order to determine whether Cbfa1expression, induced by ectopic
Ihh, leads to differentiation of mature osteoblasts, we next examined the
expression of osteocalcin. In the tibia of E16.5 wild-type embryos, a
morphologically identifiable bone collar is only present at the level of the
diaphysis, and osteocalcin is only detected in the diaphyseal bone
collar (Fig. 7G). No
osteocalcin expression is detected in the perichondrium at the level
of hypertrophic cartilage expressing Col1(X)
(Fig. 7E). In Col2-Gal4;
UAS-Ihh littermates however, bone is readily identifiable at a level
proximal to the hypertrophic zone, and osteocalcin expression is
evident in cells associated with the precocious bone collar
(Fig. 7F,H). Interestingly,
unlike the up-regulation of Cbfa1, the precocious bone collar does
not extend throughout the perichondrium (compare
Fig. 7D and H). Thus ectopic
Ihh promotes bone collar formation in the long bone but it alone is not
sufficient to induce osteogenesis throughout the perichondrium.
To examine the relationship between Hedgehog signaling and other osteogenic
factors such as BMPs, we performed cell culture studies using a mouse limb bud
cell line MLB13MYC clone 17 that is known to undergo osteoblast
differentiation upon treatment with BMPs
(Rosen et al., 1994). BMP2
induces expression of both Cbfa1 and osteocalcin after 2
days (Fig. 8, lane 2).
Interestingly, Bmp2 also induces expression of Ihh and up-regulates
Ptc1 (Fig. 8, lane 2).
The N-terminal fragment of Shh (N-Shh, an established surrogate for Ihh)
however, does not itself induce either Cbfa1 or osteocalcin
in this cell line, even though it greatly upregulates Ptc1
(Fig. 8, lane 3). Importantly,
a blocking antibody against Shh and Ihh, 5E1
(Fig. 8, lane 7) inhibits the
induction of osteocalcin and Cbfa1 by Bmp2 either completely
or partially (Fig. 8, lane 4)
whereas an unrelated monoclonal IgG has no effect
(Fig. 8, lane 5). The block of
BMP2-induced Cbfa1 and osteocalcin expression is specific as
the induction of Id1 (also known as Idb1), a target of BMP
signaling (Katagiri et al.,
1994
), is not inhibited by 5E1
(Fig. 6A, lane 4) whereas
noggin, a potent inhibitor of BMPs, blocks Id1 expression
(Fig. 8, lane 6). These results
indicate that Hh signaling is required for Bmp2-induced osteoblast
differentiation in MLB13MYC clone 17 cells, and Hh and BMP pathways cooperate
to establish the mature osteoblast phenotype.
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Discussion |
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Interestingly in Cre3; Smon/c and Cre10;
Smon/c embryos, ectopic chondrocytes separate the
bone collar from the underlying cartilage and bone marrow cavity. It is
possible that this simply results from compromised cartilage removal due to
the lack of Ihh signaling in chondrocytes, however, we consider this unlikely
since Cre15; Smon/c embryos are normal in this
regard. Alternatively, the ectopic chondrocytes could be derived from
perichondrial cells that fail to undergo osteoblast differentiation in the
absence of a direct Ihh input. This interpretation is consistent with data
from the chimeric embryos in which Smon/n
perichondrial cells also differentiate into chondrocytes. Therefore, certain
progenitor cells residing within the perichondrium are capable of developing
into either osteoblasts or chondrocytes, depending upon the presence or
absence of Ihh signaling. Interestingly in this regard, in the absence of
Osx the perichondrium also undergoes chondrocyte differentiation
(Nakashima et al., 2002)
whereas loss of Sox9 activity in the cranial neural crest cells appear to
result in ectopic osteoblast development
(Mori-Akiyama et al., 2003
).
It is not known whether Ihh, Osx and Sox9 interact to regulate cell fate. The
molecular identity of the bipotential progenitor cells in the perichondrium
also is not clear at present, but they evidently express collagen type II
transiently since the Col
1(II)-Cre transgene in the
Cre3 and Cre10 lines is expressed in both lineages. The
transgene in the Cre15 line is expressed later during development and
apparently mostly in the chondrocyte lineage. Although it remains possible
that the transient activity of the Col
1(II) promoter
in the perichondrium of the transgenic lines does not faithfully represent
expression of the endogenous gene, another
Col
1(II)-Cre transgenic line independently developed
using a longer version of the Col
1(II) regulatory
sequences (Ovchinnikov et al.,
2000
) also expresses Cre in both the chondrocyte and the
osteoblast lineages, and generates a similar bone phenotype when crossed to
the Smo conditional allele (data not shown).
Our experiments also demonstrate that Hh signaling is required for development of the primary spongiosa. Both Cre3; Smon/c and Cre10; Smon/c but not Cre15; Smon/c embryos fail to develop primary spongiosa. Since Cre3 and Cre10 remove Smo from the perichondrium whereas Cre15 does not, it is likely that osteoblasts forming the primary spongiosa originate in the perichondrium. In addition, the long bones of the Cre3; Smon/c and Cre10; Smon/c embryos also lack certain morphologically distinct cells at the cartilage-marrow junction. These cells are probably in the osteoblast lineage as they physically associate with nascent bone spicules. Of note, within this group of cells a subpopulation immediately adjacent to the last row of hypertrophic chondrocytes expresses high levels of Ptc1, indicative of a robust response to Ihh. Therefore, it is possible that during normal development of the primary spongiosa, osteoprogenitors derived from the perichondrium undergo osteoblast differentiation at the chondro-osseous junction upon sensing Ihh. In the mutant embryos, the progenitor cells devoid of Smo do not respond to Ihh and thus fail to become osteoblasts. Alternatively, the perichondrium-derived osteoprogenitor cells in the absence of Ihh signaling could fail to migrate to the potential chondro-osseous junction. The present study does not distinguish between these possibilities.
Interestingly, although Hh signaling is indispensable for osteogenesis in the limb, it is apparently not necessary for the development of certain other bones such as the mandible. The mandible is of neural crest origin and undergoes intramembranous ossification. It remains to be determined whether all bones of this origin develop independent of Hh signaling.
Hh signaling appears to cooperate with other molecules to induce osteoblast
differentiation. Although Ihh, when ectopically expressed in all chondrocytes,
advances the frontier of the bone collar, it does not induce bone throughout
the perichondrium. A similar result was observed in studies in which chimeric
growth plates containing both wild-type and PTH/PTHrP receptor null
chondrocytes were generated (Chung et al.,
1998; Chung et al.,
2001
). Bone collar extended up toward the epiphysis when
Ihh was expressed by mutant chondrocytes near the epiphysis, but not
when the chondrocytes were mutant for both the PTH/PTHrP receptor and
Ihh. These results suggest that other signals must combine with Ihh
to generate mature osteoblasts. The studies using MLB13MYC clone 17 cells
suggest that BMPs require concomitant Ihh signaling to induce osteoblasts in
cells derived from the limb bud. In addition to BMPs, Wnt signaling has
recently been implicated in osteogenesis
(Gong et al., 2001
;
Kato et al., 2002
;
Little et al., 2002
). It
remains to be determined whether the Hh, BMP and Wnt pathways also interact in
normal osteoblast differentiation.
In summary, our data support a model in which Ihh acts on a bipotential osteochondroprogenitor cell that is located within the perichondrial region to promote osteoblast specification while inhibiting the alternative chondrocyte pathway.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Internal Medicine, Department of Molecular
Biology and Pharmacology, Washington University Medical School, St. Louis,
MO
Present address: Department of Bone and Cartilage Regenerative Medicine,
University of Tokyo School of Medicine, Tokyo, Japan
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Byrd, N., Becker, S., Maye, P., Narasimhaiah, R., St-Jacques, B., Zhang, X., McMahon, J., McMahon, A. and Grabel, L. (2002). Hedgehog is required for murine yolk sac angiogenesis. Development 129,361 -372.[Medline]
Caplan, A. I. and Pechak, D. G. (1987). The cellular and molecular embryology of bone formation. In Bone and Mineral Research vol. 5 (ed. W. A. Peck), pp. 117-183. New York, NY: Elsevier.
Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering and transducing Hedgehog. Cell 87,553 -563.[Medline]
Chung, U. I., Lanske, B., Lee, K., Li, E. and Kronenberg, H.
(1998). The parathyroid hormone/parathyroid hormone-related
peptide receptor coordinates endochondral bone development by directly
controlling chondrocyte differentiation. Proc. Natl. Acad. Sci.
USA 95,13030
-13035.
Chung, U. I., Schipani, E., McMahon, A. P. and Kronenberg, H.
M. (2001). Indian hedgehog couples chondrogenesis to
osteogenesis in endochondral bone development. J. Clin.
Invest. 107,295
-304.
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[Medline]
Gerber, H. P. and Ferrara, N. (2000). Angiogenesis and bone growth. Trends Cardiovasc. Med. 10,223 -228.[CrossRef][Medline]
Gong, Y., Slee, R. B., Fukai, N., Rawadi, G., Roman-Roman, S., Reginato, A. M., Wang, H., Cundy, T., Glorieux, F. H., Lev, D. et al. (2001). LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107,513 -523.[Medline]
Hinchcliffe, J. R. and Johnson, D. R. (1990). The Development of the Vertebrate Limb. Oxford, UK: Clarendon Press.
Hogan, B. L. (1996). Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432-438.[CrossRef][Medline]
Karp, S. J., Schipani, E., St-Jacques, B., Hunzelman, J.,
Kronenberg, H. and McMahon, A. P. (2000). Indian hedgehog
coordinates endochondral bone growth and morphogenesis via parathyroid
hormone-related protein-dependent and -independent pathways.
Development 127,543
-548.
Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994). Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127,1755 -1766.[Abstract]
Kato, M., Patel, M. S., Levasseur, R., Lobov, I., Chang, B. H.,
Glass, D. A., 2nd, Hartmann, C., Li, L., Hwang, T. H., Brayton, C. F. et
al. (2002). Cbfa1-independent decrease in osteoblast
proliferation, osteopenia, and persistent embryonic eye vascularization in
mice deficient in Lrp5, a Wnt coreceptor. J. Cell
Biol. 157,303
-314.
Lanske, B., Karaplis, A. C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., Karperien, M., Defize, L. H., Ho, C., Mulligan, R. C. et al. (1996). PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273,663 -666.[Abstract]
Lee, K., Deeds, J. D. and Segre, G. V. (1995). Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 136,453 -463.[Abstract]
Little, R. D., Carulli, J. P., Del Mastro, R. G., Dupuis, J., Osborne, M., Folz, C., Manning, S. P., Swain, P. M., Zhao, S. C., Eustace, B. et al., (2002). A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am. J. Hum. Genet. 70,11 -19.[CrossRef][Medline]
Lewis, P. M., Dunn, M. P., McMahon, J. A., Logan, M., Martin, J. F., St-Jacques, B. and McMahon, A. P. (2001). Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105,599 -612.[CrossRef][Medline]
Long, F., Zhang, X. M., Karp, S., Yang, Y. and McMahon, A. P. (2001). Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development 128,5099 -5108.[Medline]
McLeod, M. J. (1980). Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22,299 -301.[Medline]
McMahon, A. P., Ingham, P. W. and Tabin, C. J. (2003). Developmental roles and clinical significance of hedgehog signaling. Curr. Top. Dev. Biol. 53, 1-114.[Medline]
Mori-Akiyama, Y., Akiyama, H., Rowitch, D. H. and de
Crombrugghe, B. (2003). Sox9 is required for determination of
the chondrogenic cell lineage in the cranial neural crest. Proc.
Natl. Acad. Sci. USA 100,9360
-9365.
Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J. M., Behringer, R. R. and de Crombrugghe, B. (2002). The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29.[Medline]
Ovchinnikov, D. A., Deng, J. M., Ogunrinu, G. and Behringer, R. R. (2000). Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis 26,145 -146.[CrossRef][Medline]
Pathi, S., Rutenberg, J. B., Johnson, R. L. and Vortkamp, A. (1999). Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation. Dev. Biol. 209,239 -253.[CrossRef][Medline]
Poole, A. R. (1991). The growth plate: Cellular physiology, cartilage assembly and mineralization. In Cartilage: Molecular aspects (ed. B. K. a. N. S. A. Hall), pp.179 -211. Boca Raton, FL: CRC Press.
Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81,445 -455.[Medline]
Rosen, V., Nove, J., Song, J. J., Thies, R. S., Cox, K. and Wozney, J. M. (1994). Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes. J. Bone Miner. Res. 9,1759 -1768.[Medline]
Rossert, J., Eberspaecher, H. and de Crombrugghe, B. (1995). Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J. Cell Biol. 129,1421 -1432.[Abstract]
St-Jacques, B., Hammerschmidt, M. and McMahon, A. P.
(1999). Indian hedgehog signaling regulates proliferation and
differentiation of chondrocytes and is essential for bone formation.
Genes Dev. 13,2072
-2086.
Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg, H. M. and Tabin, C. J. (1996). Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273,613 -622.[Abstract]
Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M. and Wang, E. A. (1988). Novel regulators of bone formation: molecular clones and activities. Science 242,1528 -1534.[Medline]
Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A.,
Kerr, W. G. and Soriano, P. (1997). Disruption of overlapping
transcripts in the ROSA beta geo 26 gene trap strain leads to widespread
expression of beta-galactosidase in mouse embryos and hematopoietic cells.
Proc. Natl. Acad. Sci. USA
94,3789
-3794.
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