First published online 23 April 2008
doi: 10.1242/dev.018044
Development 135, 1947-1956 (2008)
Published by The Company of Biologists 2008
Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy
Kinglun Kingston Mak1,
Henry M. Kronenberg2,
Pao-Tien Chuang3,
Susan Mackem4 and
Yingzi Yang1,*
1 Genetic Disease Research Branch, National Human Genome Research Institute, 49
Convent Drive, MSC 4472, Bethesda, MD 20892, USA.
2 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA.
3 Cardiovascular Research Institute, University of California, San Francisco, CA
94143, USA.
4 Laboratory of Pathology, National Cancer Institute, 9000 Rockville Pike,
Bethesda, MD 20892-1500, USA.
View larger version (91K):
[in this window]
[in a new window]
|
Fig. 1. Hedgehog signaling promotes chondrocyte hypertrophy in the absence of
PTHrP signaling. (A,B) Forelimbs of
PTHrP-/- or wild-type E14.5 embryos cultured for 36 hours
in the presence or absence of recombinant Shh protein or cyclopamine. Treated
limbs were compared with untreated contralateral control ones. Serial sections
of the proximal humerus of the cultured limbs were hybridized with
35S riboprobes of Ihh (A) or Col10a1 (B). The
distances from the articular end to the Ihh/Col10a1 domain
in the untreated wild-type and PTHrP-/- embryos are
indicated by orange lines. This distance was increased in the Shh-treated
wild-type humerus but reduced in the Shh-treated PTHrP-/-
humerus (white lines) compared with the untreated contralateral controls. This
distance was reduced in the cyclopamine-treated wild type, but increased in
the PTHrP-/- humerus (green lines) compared with the
untreated controlateral controls. (C) Statistical analysis (paired
Student's t-test) of the distance from articular chondrocytes to the
hypertrophic zone between treated and untreated contralateral limbs. Numbers
(n) of analyzed limbs are indicated. (D) Serial sections of
E14.5 distal humeri of the indicated genotypes stained with Safranin O (upper
panel) and hybridized with a 35S Col10a1 riboprobe (lower
panel) to detect chondrocyte hypertrophy. The distance from the articular end
to the Col10a1 expression domain (orange lines) in the
PTHrP-/- embryos was reduced compared with that in the
wild-type embryos. This distance in the
PTHrP-/-;UAS-cIhh;Col2a1-Gal4 double mutant was further
reduced compared with that of the PTHrP-/- embryos.
|
|
View larger version (100K):
[in this window]
[in a new window]
|
Fig. 2. Cell autonomous upregulation of Hh signaling in the absence of
PTHrP accelerates chondrocyte hypertrophy. (A) Skeletal
preparation of embryos at E14.5. Alizarin Red stains mineralized cartilage and
bone tissues; Alcian Blue stains unmineralized cartilage. A higher
magnification view of the forelimb is shown in the lower panel. S, scapula; H,
humerus; R, radius; U, ulna. (B) Serial sections of distal humerus were
stained with Safranin O and hybridized with indicated 35S labeled
riboprobes. The boxed articular and columnar chondrocytes regions are shown at
higher magnification in the panels below. Columnar chondrocytes are indicated
by yellow brackets and arrows. Both Ptch1c/-;Col2a1-Cre
and PTHrP-/-;Ptch1c/-;Col2a1-Cre mutants show
strong upregulation of Gli1 and Hip1, downstream target
genes of Hh signaling, in the perichondrium and synovial joint. The
Col2a1-expressing region (white line) is reduced and the
Col10a1-expressing domain (yellow line) is closer to the joint in the
PTHrP-/-;Ptch1c/-;Col2a1-Cre mutant.
|
|
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3. Cell proliferation is similarly reduced in PTHrP-/-
and PTHrP-/-;Ptch1c/-;Col2a1-Cre
mutant cartilage. (A) Comparison of BrdU-labeled chondrocytes in
the cartilage of different mutants and in the wild-type control. Boxed regions
are shown at higher magnification in the lower panel. The highly proliferating
columnar chondrocytes (bracket) were reduced in the
PTHrP-/-;Ptch1c/-;Col2a1-Cre mutant. (B)
Percentage of BrdU-labeled chondrocytes in the columnar regions (circled in
A), calculated from three independent samples to get the mean±s.d.
Student's t-test, P<0.05.
|
|
View larger version (91K):
[in this window]
[in a new window]
|
Fig. 4. Removal of Hh signaling delays chondrocyte hypertrophy in the absence of
PTHrP. (A) Skeletal preparation of E15.5 embryos.
Hindlimbs are shown at higher magnifications in the lower panel. (B)
Serial sections of tibia were stained with Safranin O and hybridized with
35S labeled Ihh and Col10a1 riboprobes.
Smoc/c;Col2a1-Cre mutant tibia showed a slight delay in
chondrocyte hypertrophy compared with that of wild-type embryos.
PTHrP-/-;Smoc/c;Col2a1- Cre mutant tibia also
showed a delay of chondrocyte hypertrophy, as compared with that of the
PTHrP-/- mutant. The proliferating chondrocyte region is
indicated by the yellow line; the hypertrophic region is indicated by the
white line. Fe, femur; T, tibia; Fi, fibula.
|
|
View larger version (52K):
[in this window]
[in a new window]
|
Fig. 5. Hedgehog signaling activates downstream targets of canonical Wnt
signaling both in vivo and in vitro. (A) Serial sections of E14.5
distal humerus were stained by Safranin O and hybridized with a 35S
labeled Lef1 probe. Lef1 expression was strongly upregulated
in the cartilage of Ptch1c/-; Col2a1-Cre mutants.
(B,C) Dual luciferase assay of primary chondrocytes isolated
from wild-type newborn pups. Primary chondrocytes were nucleofected with
Topflash reporter vectors as a read out for canonical Wnt signaling.
Recombinant Shh or Dkk1 protein was added after serum starvation and
luciferase activity was measured 24 hours later. Shh treatment or
Cre-adenovirus infection of the Ptch1c/c primary
chondrocytes activated TOPFLASH activity. Such activation was diminished by
Dkk1. (D,E) Quantitative RT-PCR was performed using RNA isolated
from primary chondrocytes. Both Axin2 and Lef1 were
significantly increased in Shh-treated primary chondrocytes or
Cre-adenovirus-infected Ptch1c/c primary chondrocytes
compared with untreated samples. Dkk1 treatment blocked the effect of Hh
signaling. (F) Immunohistochemistry of E15.5 limb sections (distal
ulna) with antibodies against phospho-Smad1, 5 and 8. More
phospho-Smad-positive cells were found in the cartilage of the
Ptch1c/-;Col2a1-Cre mutant embryos. Boxed region of
columnar/prehypertrophic chondrocytes is shown at a higher magnification on
the right-hand side. (G) Statistical analysis of phospho-Smad-positive
cells in the boxed region of the cartilage. Three samples in the boxed area
were counted, and the mean±s.d. are shown. Student's
t-test, P<0.05.
|
|
View larger version (87K):
[in this window]
[in a new window]
|
Fig. 6. Hedgehog signaling accelerates chondrocyte hypertrophy in postnatal
cartilage. (A) Skeletal preparations of P15 forelimb and hindlimb
of the Ptch1c/c;Col2a1-CreER and wild-type mouse.
Unmineralized cartilage in the hand, shoulder and knee joints and the scapula
was reduced in the mutant, as shown at high magnification. (Ba-d)
Serial sections of distal femur of P12 mice were stained with Safranin O and
used for immunohistochemistry to detect Col2a1 expression. There were more
hypertrophic chondrocytes with reduced Col2a1 expression in the
Ptch1c/c;Col2a1-CreER mouse. (Be-j) Sections of
proximal humeri from one-year-old mice were stained with Safranin O. Boxed
regions in e and f are shown at higher magnifications in g,h. Cartilage lining
was thinner in the Ptch1c/c;Col2a1-CreER mouse (arrow).
(i,j) Higher magnification of articular cartilage of the proximal humerus.
Joint cartilage in the Ptch1c/c;Col2a1-CreER mouse was
significantly reduced (double-headed arrows). (Ca-d) Sections of
proximal humeri from 14-month-old mice were stained with Safranin O. Boxed
regions in a and b are shown at higher magnifications in c,d. Cartilage lining
was thicker in the Smoc/c;Col2a1-CreER mouse (arrow).
(Ce,f) Higher magnification of articular cartilage of the
proximal humerus. Joint cartilage in the
Smoc/c;Col2a1-CreER mouse was significantly thicker
(double-headed arrows).
|
|
View larger version (51K):
[in this window]
[in a new window]
|
Fig. 7. Model of the PTHrP-dependent and PTHrP-independent
function of Ihh signaling in chondrocyte differentiation. The morphology
of a developing long bone is schematically shown. Chondrocytes proliferate and
differentiate sequentially along the longitudinal axis. The length of the
columnar chondrocyte region is determined by the rate of periarticular
chondrocyte differentiation, columnar chondrocyte proliferation and
hypertrophy. Ihh expression is shown in red. PTHrP
expression is shown in yellow. Ihh acts through PTHrP to
inhibit chondrocyte hypertrophy. Ihh acts independently of
PTHrP to control chondrocyte proliferation, periarticular chondrocyte
differentiation and osteoblast differentiation. Here, we show that
Ihh also promotes chondrocyte hypertrophy independently of
PTHrP and that this function is likely to be mediated by Bmp and
Wnt/β-catenin signaling.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
