Develop. Growth Differ.
expression in relation to chondrocyte proliferation during
mouse bone development
Helen E. MacLean
and Henry M. Kronenberg*
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
We have developed a useful approach to examine the pattern of gene expression in comparison to cell
proliferation, using double in situ hybridization and immunofluorescence. Using this system, we examined the
expression of Indian hedgehog ( Ihh) and PTH/PTHrP receptor
proliferation during embryonic mouse bone development. Both genes are expressed strongly in pre-
hypertrophic and early hypertrophic chondrocytes, and there is a strong correlation between upregulation of
Ihh and PPR expression and chondrocyte cell cycle arrest. At embryonic day (E14.5),
lation begins in the columnar chondrocytes just prior to cell cycle exit, but at later time points expression is only
observed in the postproliferative region. In contrast, Ihh
columnar proliferating chondrocytes at all stages. This study provides further evidence that in the developing
growth plate, cell cycle exit and upregulation of
) mRNA in relation to chondrocyte
mRNA expression overlaps slightly with the region of
mRNA expression are coupled.
chondrocyte, differentiation, Indian hedgehog, proliferation, PTH/PTHrP receptor.
Endochondral bone development requires the careful
coordination of chondrocyte proliferation and differ-
entiation, to allow growth of the cartilage mold and
subsequent bone formation. During their proliferative
phase, chondrocytes first appear as small rounded
cells, which flatten to form proliferating columns. The
columnar chondrocytes then stop proliferating, and
further differentiate into prehypertrophic and hyper-
trophic chondrocytes. Hypertrophic chondrocytes are
mineralized, and vascular invasion occurs, allowing
the replacement of mineralized cartilage with bone
and bone marrow (Karsenty & Wagner 2002). Para-
thyroid hormone-related peptide (PTHrP) and Indian
hedgehog (Ihh) are two of the major regulators of
chondrocyte proliferation, which coordinate the rate
of chondrocyte proliferation and the timing of the
decision to exit the proliferative pool and differentiate
et al. 1996; Vortkamp
PTHrP is produced by the perichondrial cells and
early proliferative chondrocytes at the ends of the
cartilage mold (Lee
the G protein-coupled PTH/PTHrP receptor (PPR) to
maintain chondrocytes in the proliferative pool. The
major action of PTHrP is directly on proliferative
PPR mRNA expression can be detected.
PPR expression is dramatically upregulated
in prehypertrophic chondrocytes, and PTHrP has
been demonstrated by immunohistochemistry to be
present in these cells (Lee
thought to be bound to the PPR. Chondrocyte cell
cycle exit occurs when the degree of PTHrP signaling
in columnar chondrocytes is diminished, and cells
differentiate into prehypertrophic
which synthesize Ihh. Ihh acts through its receptor,
Patched-1, to cause the production of PTHrP by the
perichondrial cells, and also to directly stimulate
chondrocyte proliferation (St-Jacques
et al . 2000; Long et al
Ihh regulate the size of the proliferative chondrocyte
pool, by controlling proliferation rate and the site at
which chondrocytes leave the proliferative pool. The
precise relationship between the site at which cell
cycle exit occurs and at which expression of
PPR is upregulated has not been examined. There-
fore, in this study we have further refined the expres-
sion patterns of
expression in relation to chondrocyte proliferation,
during embryonic bone development.
. 1996), and acts through
. 1998), where only low
. 1996), where it is
. 2001). Thus, PTHrP and
by examining their
*Author to whom all correspondence should be addressed.
†Current address: Department of Medicine (AH/NH), Univer-
sity of Melbourne, Parkville, VIC, 3052, Australia
Received 27 October 2004; revised 30 November 2004;
accepted 3 December 2004.
60H. E. MacLean and H. M. Kronenberg
Materials and Methods
All animal studies were undertaken according to
institutional guidelines. C57BL6 mice were time-
mated, with embryonic day 0.5 (E0.5) defined as noon
on the day a vaginal plug was observed.
bromodeoxyuridine (BrdU) labeling of proliferating
cells was performed prior to harvesting embryos, as
et al . 2003).
In situ hybridization for Ihh
was first performed on sections of tibiae, as previ-
ously described (MacLean
following development of emulsion, no counter-
staining was performed. Instead, immunofluorescent
detection of BrdU was performed using fluorescein-
conjugated anti-BrdU (Roche Diagnostics, Basel,
Switzerland), as follows. Slides were rehydrated in
phosphate-buffered saline (PBS) for 15 min, quen-
ched in 3% H
for 10 min, washed in PBS three
times for 2 min each, and then treated with blocking
reagent from the tyramide signal amplification
(TSA)-biotin amplification system (Perkin Elmer Life
Sciences, Boston, MA, USA) for 30 min at room
temperature. Slides were then incubated 1 h at
room temperature with anti-BrdU-fluorescein (Roche),
diluted 1:1 in blocking reagent. Slides were washed
three times in PBS for 5 min each, then mounted in
Vectashield with 4
(Vector Laboratories, Burlingame, CA, USA), diluted
1:1 in Tris buffered saline (TBS) pH 9.0. Microscopy
was performed using dark field and fluorescein filters,
and images were merged using Adobe Photoshop.
All analyses were performed on 2–3 sections per
developmental stage. The point at which chondro-
cytes exit the proliferative pool was determined from
the BrdU immunofluorescence, by identifying the
position in the tibia beyond which no BrdU-positive
chondrocytes were present. Tibial sections from
double immunofluorescence and
were also compared with adjacent or developmental
age-matched sections stained with hematoxylin and
eosin, to confirm the morphology of the growth plate.
Prehypertrophic chondrocytes were defined as the
chondrocytes that have ceased prolifeation and that
express prehypertrophic markers including
PPR . Hypertrophic chondrocytes were identified
morphologically and by expression of the hyper-
et al . 2003).
. 2003), except that
, as previously described
Results and Discussion
To examine the relationship between
mRNA expression and chondrocyte proliferation, we
developed a system of double
and BrdU immunofluorescence, using sections from
embryonic mouse tibiae. Embryos were labeled
in utero with BrdU to identify proliferating chondro-
cytes, and sections of tibiae from E14.5–E18.5 mice
were examined to localize
expression in relation to the BrdU-positive, prolifer-
ative chondrocyte region, using
and immunofluorescence on single sections. Captur-
ing images from this analysis using dark field
in situ hybridization) and fluorescence
microscopy (BrdU immunofluorescence), and merg-
ing the images, allowed the side by side comparison
of gene expression in relation to cell proliferation.
At E14.5, the chondrocyte proliferation rate was high
at the ends of the tibiae, evidenced by the large
number of BrdU-positive, green cells in this region
(Fig. 1A,B). In the center of the tibia, prehypertrophic
and early hypertrophic chondrocytes expressed both
Ihh and PPR mRNA (Fig. 1A,B). When expression of
these genes was compared to the timing of cell cycle
exit at E14.5, expression of
the region of proliferating chondrocytes than did
expression (Fig. 1B vs. 1A). Thus, the upregulation of
Ihh expression coincided almost exactly with the
cessation of proliferation, whereas there was a more
gradual increase in
PPR expression that started in the
columnar proliferating chondrocytes, and increased in
intensity in the postproliferative region.
At E15.5, the tibia contains mature hypertrophic
chondrocytes in the center, so the expression domains
Ihh and PPR were reduced. The gradient of
mRNA upregulation at E15.5 was sharper than the
expression pattern observed at E14.5, so that there
was a striking increase in
ponding almost exactly with the point at which
chondrocytes stop proliferating (Fig. 1D vs 1B). This
sharp increase in
PPR mRNA expression that
coincides with cell cycle exit was also observed at
E16.5–E18.5 (Fig. 1F,H,J). The fact that
sion was detectable in the proliferating chondrocytes
at E14.5 but not at later stages may relate to the
fact that PTHrP has a greater effect on stimulating
chondrocyte proliferation rate at earlier developmental
time points than at later stages (Karp
higher expression of
PPR mRNA in E14.5 proliferating
chondrocytes could potentially make these cells more
responsive to the stimulatory actions of PTHrP than at
later time points.
In contrast, at E15.5 and later time points the
Ihh mRNA expression was more
gradual than that of
onwards, there was some overlap between where
expression commenced and the region containing
overlapped more with
. 2000). The
expression. From E15.5
expression in chondrocytes 61
columnar proliferating chondrocytes (Fig. 1C,E,G,I),
although expression of
postproliferative prehypertrophic chondrocytes. Thus,
PPR mRNA expression was detectable prior
Ihh expression, in the region of proliferating
chondrocytes, with maximal intensity of both genes'
expression occurring in the postproliferative cells. In
contrast, at E15.5 and later stages, low levels of
mRNA expression occurred in the final layers of
was still higher in the
the proliferative chondrocytes, whereas
expression was only observed in the region where
cell cycle exit had occurred. At all stages,
PPR expression was highest in the postproliferative
chondrocytes, with a strong correlation between areas
where chondrocytes had stopped proliferating and
Ihh and PPR mRNA was expressed strongly.
The expression of
Ihh by prehypertrophic and early
hypertrophic chondrocytes allows the formation of a
examined for expression of either
in situ hybridization (dark-field image) and BrdU immunofluorescence, and the two images merged, from E14.5 (A,B), E15.5 (C,D),
E16.5 (E,F), E17.5 (G,H) and E18.5 (I,J) tibiae. The orange dashed lines indicate the point where chondrocytes exit the proliferative
pool, determined as described in the Materials and Methods. H&E indicates E14.5–E18.5 serial sections stained with hematoxylin and
eosin, to identify the morphology of the growth plate.
hybridization and bromodeoxyuridine (BrdU) immunofluorescence on mouse tibial sections. Each section was
Ihh mRNA (A,C,E,G,I) or PPR mRNA (B, D, F, H, J), and for BrdU incorporation. Panels show separate
62 H. E. MacLean and H. M. Kronenberg
negative feedback loop with PTHrP, to regulate the
distance from the articular surface where hyper-
trophic differentiation occurs (Lanske
et al . 1996). The reason
increased in these chondrocyte populations is less
clear. Although PTHrP acts directly on proliferating
et al . 1998), it is also thought to
act on prehypertrophic cells to modulate the rate of
transition from proliferation to differentiation (Huang
et al. 2001). PTHrP may also play further roles in
regulating the differentiation of hypertrophic chondro-
cytes through Runx2 (Guo
PTHrP is produced at the ends of the cartilage
mold, the concentration of PTHrP reaching the pre-
hypertrophic chondrocytes in the tibia may be low,
and upregulation of PPR expression may be required
to increase the sensitivity of these cells to PTHrP. The
increased PPR expression in the prehypertrophic
chondrocytes may also potentially sharpen the
gradient of PTHrP action by creating a barrier to
prevent PTHrP reaching the hypertrophic chondro-
The factors regulating the increased Ihh and PPR
mRNA expression in prehypertrophic chondrocytes
have not been fully elucidated. Ihh expression is
partly suppressed by fibroblast growth factor (FGF)
signaling (Liu et al. 2002; Minina et al. 2002;
Ohbayashi et al. 2002) and activated by Runx2 and
Runx3 (Takeda et al. 2001; Yoshida et al. 2004), and
Sox9 may also upregulate Ihh and PPR expression
(Akiyama et al. 2002). The fact that the highest
expression of both Ihh and PPR occurs in chondro-
cytes of the tibia after cell cycle exit suggests that
these processes are coupled. The pRb-related pocket
proteins p107 and p130 are required for normal cell
cycle exit in chondrocytes (Cobrinik et al. 1996).
Runx2 mRNA expression is decreased in p107/p130-
null chondrocytes, and only occurs in cells under-
going proliferative arrest (Rossi et al. 2002). Therefore,
the upregulation of Runx2 in postproliferative chondro-
cytes may lead to the upregulation of Ihh expression
(Yoshida et al. 2004), providing a mechanism to
couple cell cycle exit with terminal differentiation.
Conversely, the fact that we observed differences in
the border between exit of proliferation and upregu-
lation of Ihh and PPR mRNA expression at different
developmental stages, suggests that other factors are
also regulating the expression of these genes. For
example, at E14.5 PPR mRNA clearly overlaps with the
proliferating chondrocytes in the tibia, whereas at later
stages it does not, indicating that at earlier stages
the exit of proliferation and PPR expression are more
loosely coupled. Similarly, the upregulation of Ihh
mRNA expression precedes cell cycle exit at E15.5
et al. 2003). Because
and later, further illustrating that exit of proliferation is
not an absolute requirement for upregulation of Ihh
and PPR gene expression.
In summary, using double in situ hybridization and
immunofluorescence, we have carried out the first
side by side comparison of Ihh and PPR mRNA
expression in relation to chondrocyte proliferation
during embryonic bone development. We have
demonstrated that in general there is a strong
correlation between upregulation of both Ihh and
PPR expression and chondrocyte cell cycle arrest.
At E14.5, PPR mRNA upregulation begins in the
columnar chondrocytes just prior to cell cycle exit, but
at later time points expression is only observed in the
postproliferative region. In contrast, Ihh mRNA expres-
sion overlaps slightly with the region of columnar
proliferating chondrocytes at all stages. This study
demonstrates a useful approach to examining the
pattern of gene expression in comparison to cell
proliferation, and provides further evidence that in the
developing growth plate, cell cycle exit and upregu-
lation of Ihh and PPR mRNA expression are coupled.
We thank Janet Saxton and Manny Armijos for histo-
logical assistance. This study was supported by
National Institutes of Health grant DK-56246 to H.M.K;
H.E.M. was supported by an NHMRC (Australia) C.J.
Martin Fellowship #987029 and a Massachusetts
General Hospital Fund for Medical Discovery
Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A., de
Crombrugghe, B. 2002. The transcription factor Sox9 has
essential roles in successive steps of the chondrocyte
differentiation pathway and is required for expression of
Sox5 and Sox6. Genes Dev. 16, 2813–2828.
Chung, U. I., Lanske, B., Lee, K., Li, E., Kronenberg, H. 1998.
The parathyroid hormone/parathyroid hormone-related
peptide receptor coordinates endochondral bone develop-
ment by directly controlling chondrocyte differentiation.
Proc. Natl Acad. Sci. USA. 95, 13030–13035.
Cobrinik, D., Lee, M. H., Hannon, G. et al. 1996. Shared role of
the pRB-related p130 and p107 proteins in limb develop-
ment. Genes Dev. 10, 1633–1644.
Guo, J., Chung, U., Szczepanik, A., Yang, D., Bringhurst, F. R.,
Kronenberg, H. M. 2003. PTH/PTHrP receptor delays
chondrocyte hypertrophy via both Runx2-dependent and
independent pathways. J. Bone Miner. Res. 18 (Suppl. 1),
Huang, W., Chung, U. I., Kronenberg, H. M., de Crombrugghe,
B. 2001. The chondrogenic transcription factor Sox9 is a
target of signaling by the parathyroid hormone-related
peptide in the growth plate of endochondral bones. Proc.
Natl Acad. Sci. USA. 98, 160–165.
Ihh and PPR expression in chondrocytes 63
Karp, S. J., Schipani, E., St-Jacques, B., Hunzelman, J.,
Kronenberg, H., McMahon, A. P. 2000. Indian hedgehog
coordinates endochondral bone growth and morpho-
genesis via parathyroid hormone related-protein-dependent
and -independent pathways. Development 127, 543–548.
Karsenty, G., Wagner, E. F. 2002. Reaching a genetic and
molecular understanding of skeletal development. Dev. Cell
Lanske, B., Karaplis, A. C., Lee, K. et al. 1996. PTH/PTHrP
receptor in early development and Indian hedgehog-
regulated bone growth. Science 273, 663–666.
Lee, K., Lanske, B., Karaplis, A. C. et al. 1996. Parathyroid
hormone-related peptide delays terminal differentiation of
chondrocytes during endochondral bone development.
Endocrinology 137, 5109–5118.
Liu, Z., Xu, J., Colvin, J. S., Ornitz, D. M. 2002. Coordination of
chondrogenesis and osteogenesis by fibroblast growth
factor 18. Genes Dev. 16, 859–869.
Long, F., Zhang, X. M., Karp, S., Yang, Y., McMahon, A. P. 2001.
Genetic manipulation of hedgehog signaling in the endo-
chondral skeleton reveals a direct role in the regulation of
chondrocyte proliferation. Development 128, 5099–5108.
MacLean, H. E., Kim, J. I., Glimcher, M. J., Wang, J.,
Kronenberg, H. M., Glimcher, L. H. 2003. Absence of
transcription factor c-maf causes abnormal terminal
differentiation of hypertrophic
endochondral bone development. Dev. Biol. 262, 51–63.
Minina, E., Kreschel, C., Naski, M. C., Ornitz, D. M., Vortkamp,
A. 2002. Interaction of FGF, Ihh/Pthlh, and BMP signaling
integrates chondrocyte proliferation and hypertrophic
differentiation. Dev. Cell 3, 439–449.
Ohbayashi, N., Shibayama, M., Kurotaki, Y. et al. 2002. FGF18
is required for normal cell proliferation and differentiation
during osteogenesis and chondrogenesis. Genes Dev. 16,
Rossi, F., MacLean, H. E., Yuan, W. et al. 2002. p107 and p130
coordinately regulate proliferation, Cbfa1 expression, and
hypertrophic differentiation during endochondral bone
development. Dev. Biol. 247, 271–285.
St-Jacques, B., Hammerschmidt, M., McMahon, A. P. 1999.
Indian hedgehog signaling regulates proliferation and
differentiation of chondrocytes and is essential for bone
formation. Genes Dev. 13, 2072–2086.
Takeda, S., Bonnamy, J. P., Owen, M. J., Ducy, P., Karsenty, G.
2001. Continuous expression of Cbfa1 in nonhypertrophic
chondrocytes uncovers its ability to induce hypertrophic
chondrocyte differentiation and partially rescues Cbfa1-
deficient mice. Genes Dev. 15, 467–481.
Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg,
H. M., Tabin, C. J. 1996. Regulation of rate of cartilage
differentiation by Indian hedgehog and PTH-related protein.
Science 273, 613–622.
Yoshida, C. A., Yamamoto, H., Fujita, T. et al. 2004. Runx2 and
Runx3 are essential for chondrocyte maturation, and Runx2
regulates limb growth through induction of Indian hedge-
hog. Genes Dev. 18, 952–963.