Haploinsufficiency of Sox9 results in defective
cartilage primordia and premature
Weimin Bi, Wendong Huang, Deanne J. Whitworth, Jian Min Deng, Zhaoping Zhang, Richard R. Behringer,
and Benoit de Crombrugghe*
Department of Molecular Genetics and Graduate Program in Genes and Development, M. D. Anderson Cancer Center, University of Texas,
Houston, TX 77030
Edited by Darwin J. Prockop, Tulane University, New Orleans, LA, and approved March 30, 2001 (received for review February 23, 2001)
dysmorphology syndrome campomelic dysplasia. Except for clini-
cal descriptions, little is known about the pathogenesis of this
disease. We have generated heterozygous Sox9 mutant mice that
phenocopy most of the skeletal abnormalities of this syndrome.
The Sox9?/?mice died perinatally with cleft palate, as well as
hypoplasia and bending of many skeletal structures derived from
cartilage precursors. In embryonic day (E)14.5 heterozygous em-
bryos, bending of radius, ulna, and tibia cartilages was already
prominent. In E12.5 heterozygotes, all skeletal elements visualized
by using Alcian blue were smaller. In addition, the overall levels of
Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type
embryos. We propose that the skeletal abnormalities observed at
later embryonic stages were caused by delayed or defective pre-
cartilaginous condensations. Furthermore, in E18.5 embryos and in
newborn heterozygotes, premature mineralization occurred in
many bones, including vertebrae and some craniofacial bones.
Because Sox9 is not expressed in the mineralized portion of the
growth plate, this premature mineralization is very likely the
consequence of allele insufficiency existing in cells of the growth
plate that express Sox9. Because the hypertrophic zone of the
heterozygous Sox9 mutants was larger than that of wild-type
mice, we propose that Sox9 also has a role in regulating the
transition to hypertrophic chondrocytes in the growth plate. De-
spite the severe hypoplasia of cartilages, the overall organization
and cellular composition of the growth plate were otherwise
normal. Our results suggest the hypothesis that two critical steps
of the chondrocyte differentiation pathway are sensitive to Sox9
dosage. First, an early step presumably at the stage of mesenchy-
mal condensation of cartilage primordia, and second, a later step
preceding the transition of chondrocytes into hypertrophic
mesenchymal cells first aggregate together to form precartilag-
inous condensations that prefigure the overall shape of future
bones. Then expression of cartilage-specific proteins is initiated,
and the cells become surrounded by abundant extracellular
matrix (1). In the growth plate of endochondral bones, chon-
drocytes become flattened and undergo a unidirectional prolif-
eration that is primarily responsible for the longitudinal growth
of bones. After these cells stop proliferating, they change their
genetic program and become hypertrophic. The extracellular
matrix surrounding the hypertrophic chondrocytes that are
closest to the metaphyses become mineralized and the cells
undergo apoptosis, leaving behind a calcified cartilaginous
matrix that is degraded subsequently and replaced by bone
Recent studies have identified SOX9 as an essential transcrip-
tion factor in chondrogenesis (3). In the absence of Sox9, there
is a complete block in chondrocyte differentiation, and the block
occurs at the stage of mesenchymal condensations. SOX9 con-
he differentiation of chondrocytes from mesenchymal cells
occurs along a multistep pathway, during which committed
tains an SRY-related hydroxymethyl glutaryl (HMG) box DNA-
binding domain and a transactivation domain rich in proline and
glutamine. During mouse embryonic development, Sox9 is ex-
pressed prominently in all chondrogenic precursor cells and in
chondrocytes throughout the deposition of cartilage-specific
matrix (4), but the expression of Sox9 is switched completely off
in hypertrophic chondrocytes (5). In addition to skeletal ele-
ments, Sox9 also is expressed in developing gonads, heart,
kidney, central nervous system, and pancreas (6, 7).
It has been suggested that the dosage of SOX9 is critical for its
normal function in humans. Mutations in a single allele of SOX9
cause a severe skeletal malformation syndrome called cam-
pomelic dysplasia (CD; refs. 4, 6, and 8). In this disease, in which
most skeletal elements derived from cartilage are affected, the
characteristic clinical features are bowing and angulation of the
tibiae and femura, hypoplastic scapulae, a missing pair of ribs,
cleft palate, and micrognathia (9, 10). Nonskeletal abnormalities
such as the absence of olfactory bulbs, dilatation of cerebral
ventricles, and a variety of cardiac and renal defects also have
been reported. In addition, about three-fourths of XY individ-
uals develop as phenotypic females or intersexes (10).
Except for clinical descriptions of the disease, little is known
about its pathogenesis. To better understand the human disease
we generated heterozygous Sox9 mutant mice. These mice die
shortly after birth and display bending of long bones and
hypoplasia of a variety of skeletal elements that strongly resem-
ble those in patients with CD. Analysis of the course of the
skeletal defects during embryonic development indicated that
the cartilage primordia were malformed. Furthermore, prema-
ture mineralization was observed in a series of bones. Our results
indicate that Sox9 haploinsufficiency is manifested at two critical
steps in the chondrocyte differentiation pathway—first, at an
early step when cartilage primordia are formed and second, at a
step preceding hypertrophic chondrocyte maturation.
Materials and Methods
Generation and Genotyping of the Sox9 Mutant Mice. Embryonic
stem cell lines containing mutations in one of the Sox9 alleles
were injected into blastocysts isolated from C57B6 mice that
were reimplanted into the uteri of pseudopregnant CD-1 mice.
Six male chimeras from two independent cell lines yielded
germline transmission. The degree of chimerism of the founder
mice varied from 70% to 95% based on agouti coat color. The
heterozygotes were identified by Southern blot analysis and
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: En, embryonic day n; CD, campomelic dysplasia; ES, embryonic stem.
*To whom reprint requests should be addressed at: Department of Molecular Genetics,
1515 Holcombe Boulevard, M. D. Anderson Cancer Center, University of Texas, Houston,
TX 77030. E-mail: firstname.lastname@example.org.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
June 5, 2001 ?
vol. 98 ?
?-galactosidase analysis. For Southern blot analysis, tail DNA
was digested with EcoRI and hybridized with a 3? external probe,
as described (3).
Yolk sac-derived genomic DNA from embryos at E15.5 or
earlier stages were genotyped by PCR. Primers specific for the
Sox9 wild-type allele were located inside the deleted region
(sense primer, 5?-TGAATCTCCTGGACCCCTTC-3? and anti-
sense primer, 5?-TGCTGGAGCCGTTGACGCG-3?). Primers
specific for the mutant allele were inside the neomycin gene
(sense primer, 5?-GCCCTGAATGAACTGCAGGACG-3? and
antisense primer, 5?-CACGGGTAGCCAACGCTATGTC-3?).
The reaction condition is 30 sec at 94°C, 1 min at 60°C, and 1 min
at 72°C for 35 cycles.
Skeletal Analysis. Embryos at E14.5, E15.5, and E18.5 were
dissected in PBS, and neonates were killed by using dry ice. The
neonates then were skinned, eviscerated, and fixed in 95%
ethanol. Skeletal preparation was performed as described (11).
Alcian blue staining of cartilaginous tissue in E12.5 and E14.5
embryos was done according to a modification of the chicken
embryo procedure (12).
Histological Analysis, in Situ Hybridization, and Immunohistochemical
Analysis. Embryos were fixed in 4% formaldehyde, dehydrated
with increasing ethanol concentration, and embedded in paraf-
fin. Serial sections of 7 ?m were stained with hematoxylin and
eosin or Alcian blue. 5-bromo-4-chloro-3-indolyl ?-D-
galactoside (X-Gal) staining and radioactive RNA in situ hy-
bridization were performed on sections as described (3). The
Col10a1 probe is a 550-bp BamHI and HindIII fragment of
mouse type X collagen cDNA.
Northern and Western Analyses. Primary chondrocytes were iso-
lated from mouse rib cartilages as described (13). Briefly, ribs
were dissected in PBS and digested with collagenase B. The
isolated chondrocytes were used directly for RNA and cell
lysates. Total RNA was prepared by RNeasy miniprep kit
(Qiagen, Chatsworth, CA). The Sox9 probe is a 300-bp fragment
corresponding to the deleted region, and the Col2a1 probe is a
405-bp HaeIII and DraI fragment from the exon corresponding
to the 3? untranslated sequence of Col2a1 RNA (14). The same
membrane was hybridized subsequently with Sox9, Col2a1, and
GAPDH probes by stripping and rehybridization. Western anal-
ysis was done with the enhanced chemiluminescence (ECL) kit
from Amersham Pharmacia. Cell lysates were prepared from
primary chondrocytes as described (15). SOX9 antibodies were
described and used at a 1:1000 dilution (16). ?-actin antibodies
(Sigma) were diluted 1:1000.
Sox9 Heterozygotes Die Shortly After Birth. To determine the role
of Sox9, we previously had generated Sox9 heterozygous
(Sox9?/?) and homozygous (Sox9?/?) mutant ES cells by ho-
mologous recombination (3). In the mutant allele of Sox9, a
300-bp deletion of part of exon 1, including the translation
initiation site and a portion of the sequence coding for the HMG
domain, was replaced with an IRES-lacZ-PGK-neo-bpA cas-
sette. No Sox9 transcripts were detected in E10.5 embryos
derived exclusively from Sox9 homozygous mutant ES cells,
indicating that the mutant allele we generated was null for Sox9
transmission of the Sox9 lacZ-neo allele was obtained for two
targeted clones. Sox9?/?mice derived from these clones exhib-
ited the same mutant phenotype. Chondrocytes from Sox9?/?
newborn mice showed a significant reduction in the expression
of both Sox9 RNA and protein compared with wild-type chon-
drocytes (Fig. 1).
All Sox9?/?mutants generated by crossing chimeric male
founders and CD-1 females died within the first 20 h after birth.
Both male and female Sox9?/?mutants were obtained. The
mutants displayed a gasping respiration and accumulated air in
more slender but had an overall length that was similar to that
of their wild-type littermates (Fig. 2 A and B). A shortened lower
jaw and frequently a crooked tail were observed in the mutant
Sox9?/?mutants had a bilateral cleft of the secondary palate
at birth. Histological analysis showed that the secondary palate
was not formed in Sox9 heterozygotes, although the two palatal
shelves were present (Fig. 2 C and D). The mutants also had a
bifurcated tongue, but tooth development was not affected. Both
Sox9 heterozygotes of different genetic backgrounds were ex-
amined. At E14.5, testes from heterozygotes of 129SvEv?C57B6
and 129SvEv?Swiss mixed backgrounds and those from a
129SvEv inbred background were histologically normal (Fig. 2 E
and F and data not shown). Expression of the Sox9 lacZ-neo
mutant allele in heterozygous mutants, detected by ?-galacto-
sidase activity, recapitulated the normal expression pattern of
Sox9 (4, 5). The expression pattern of the Sox9 mutant allele also
indicated that there was no transformation of skeletal structures,
implying that Sox9 haploinsufficiency does not affect patterning
of skeletal elements.
Skeletal Malformations in Sox9 Heterozygotes. Alcian blue and
Alizarin red S staining of Sox9?/?E18.5 and newborn mutants
revealed hypoplasia of nearly all skeletal elements derived by
degrees of bilateral and anterior bending of long bones including
the ulnae, radii, and tibiae, with the most severe bending always
observed in the ulnae (Fig. 2 G and H). The bending of the radii
occurred in the middle of the bone shaft, whereas the acute
angulation of the ulnae was more anterior in the bone shaft. The
bending was symmetrical, but the severity of bending varied
among individuals from slight bowing to obvious angulation. In
Sox9?/?E18.5 embryos, the scapulae were hypoplastic (Fig. 2H).
bent (Fig. 2J). In all heterozygotes examined, scapulae and
pelvic bones were affected symmetrically on each side of the
In Sox9?/?neonates, a small rib cage was evident that was
caused in large part by a shorter sternum (Fig. 2 K and L). The
sternebrae were also thinner and not as regular as those in
wild-type controls. The manubrium sternum of Sox9?/?mutants
was missing or exhibited anterior bending. The xiphoid process
(???) and heterozygous (???) neonates. (A) Northern analysis. Ten micro-
The same membrane then was hybridized with GAPDH as an RNA-loading
control for the quantity of RNA. (B) Western blotting with SOX9 antibody.
?-actin antibody (Sigma) was used on the same membrane as a protein-
Expression of Sox9 in primary chondrocytes isolated from wild-type
Bi et al.
June 5, 2001 ?
vol. 98 ?
no. 12 ?
was affected also, with a much smaller xiphoid cartilage (Fig. 2
K and L).
In Sox9?/?mutants, the hyoid bone, laryngeal cartilage, and
tracheal rings of the respiratory tract were thinner and stained
more weakly by Alcian blue (Fig. 2 M and N). The body of the
hyoid bone was bent in the center with the central part of the
hyoid bone missing in severely affected mutants. The malfor-
mations of the trachea in the heterozygotes indicated that the
loss of one functional Sox9 allele also affected permanent
Abnormalities in Cartilage Primordia. To understand further the
skeletal defects of Sox9?/?mutants, we examined the develop-
mental course of the skeletal malformations. Essentially all
endochondral skeletal elements of E14.5 heterozygous mutant
embryos were smaller and thinner than those in wild-type
controls (Fig. 3 A–F). At the base of the skull, the cartilage
intensely with Alcian blue. Meckel’s cartilage was interrupted
and bent toward the body midline, appearing much shorter
because of the bending (Fig. 3 I and J). This impaired Meckel’s
cartilage accounts for the smaller mandible observed in mutant
neonates. In E14.5 Sox9?/?embryos, the cartilage precursor of
scapulae were severely hypoplastic (Fig. 3 E and F and K and L);
the blades of the heterozygous scapulae consisted of two parts
that were not connected completely. Moreover, only the two
ends of the spines were present in mutants, with the major
central part missing (Fig. 3 K and L). Prominent bending and
angulation of the ulnae, radii, and tibiae were present (Fig. 3
C–H). The bending site was located outside of the mineralization
region, indicating that the bending of long bones was caused by
bending of cartilaginous bone primordia (Fig. 3 G and H).
In E15.5 Sox9 heterozygotes, formation of the sternal bars that
connect the costal cartilages at the ventral ends of the ribs was
affected. Normally at E15.5, the sternal bars are well formed and
are able to be stained by Alcian blue (Fig. 3M; ref. 17). However,
in Sox9?/?mutants, the cartilaginous sternum was shorter with
some cartilaginous elements that had not yet formed, leaving an
Alcian blue-unstained space between sternal bars (Fig. 3N). The
the skeletal preparation of wild-type (A) and heterozygous (B) newborns. (C
cleft palate in the mutants. (E and F) Transverse sections through testes of
E14.5 Sox9?/?embryos. (E) Wild-type testis (Swiss) showed seminiferous cords
(arrow) enclosing the germ cells. Fetal Leydig cells comprise the interstitium
(asterisk) between the seminiferous cords, and a tunica albuginea (arrow-
head) surrounds the testis. (F) Sox9?/?testis (129SvEv?C57B6) was histologi-
cally normal. (G–N) Dissected skeletal elements of wild-type controls (G, I, K,
and M) and Sox9?/?mutants (H, J, L, and N). (G and H) E18.5 forelimbs. The
deltoid tuberosity (Dt) of the humerus was missing in the mutants. Arrow-
Arrowhead indicates a missing manubrium sterna. (M and N) Newborn tra-
chea. Arrowhead indicates the bending of the hyoid bone in the Sox9?/?
mutants. S, scapula; H, humerus; R, radius; U, ulna; Il, ilium; Pu, pubic; Xp,
xiphoid process; Hy, hyoid bone; Tc, thyroid cartilage; Cc, cricoid cartilage; Tr,
Skeletal malformations in Sox9?/?embryos. (A and B) Lateral view of
E14.5. (A–H) Skeletal preparation of wild-type (A, C–E, and G) and Sox9?/?
and K) and Sox9?/?(J and L) embryos. Meckel’s cartilage was interrupted
(arrow) and bowing (arrowhead) in the mutants (J). (K and L) Scapulae.
Arrowheads indicate a missing spine and arrow indicates the hypoplastic
in the mutants as indicated by arrows. Me, Meckel’s cartilage; S, scapula; Sp,
spine of scapula; H, humerus; R, radius; U, ulna; F, femur; Fi, fibula; T, tibia.
Abnormalities in the cartilaginous elements in Sox9?/?embryos at
www.pnas.org?cgi?doi?10.1073?pnas.111092198Bi et al.
smaller and irregular sternal bars present at later stages were
presumably a result of delayed development of these skeletal
We concluded that the skeletal malformations in the Sox9?/?
mutants were caused by defective cartilage skeletal precursors.
Impaired Development of Precartilaginous Mesenchymes. In E12.5
Sox9?/?mutants, all precartilaginous skeletal elements visual-
ized by using Alcian blue staining exhibited reduced staining
intensity and were smaller than in wild-type controls (Fig. 4
A–D). For example, in E12.5 heterozygotes, the cartilage pri-
mordia of scapula consisted of two small pieces instead of a
single intact element, as is normally found in wild-type embryos
(Fig. 4 C and D). Histological analysis of E12.5 Sox9?/?and
wild-type embryos showed that in the heterozygotes, the chon-
droprogenitors surrounding the notochord in the vertebral pri-
mordia were still elongated mesenchymal cells with little matrix
around the cells (Fig. 4F). In contrast, in equivalent vertebral
primordia of wild-type embryos, these cells were already typical
cobblestone-like chondrogenic cells surrounded by abundant
matrix (Fig. 4E). These results suggest that despite the fact that
the domains of expression of Sox9 were unchanged in Sox9?/?
mutant embryos, the development of cartilage primordia was
delayed and was of smaller size.
Expression levels of Sox9 and Col2a1, examined by Northern
blot, were decreased when RNAs from whole E10.5 and E12.5
embryos were used (Fig. 4G). Overall, our results indicate that
correct development of cartilage primordia seems to be delayed
in heterozygotes because of dosage insufficiency of Sox9.
Premature Skeletal Mineralization in Sox9?/?Mice. Mineralization
of skeletal elements is an ordered and precisely controlled
process. In Sox9?/?neonates, premature mineralization oc-
curred in many skeletal elements including craniofacial bones
and vertebrae (Fig. 5). The heterozygotes had a similar-sized
by Alizarin red, and fewer cartilaginous regions, stained by
Alcian blue (Fig. 5 A–D). In Sox9?/?mutants, advanced min-
eralization also was observed in bony ossicles of the middle ear
(Fig. 5 A and B). Ossification centers of the interparietal and
supraoccipital bones were expanded also (Fig. 5 C and D).
The vertebrae and ribs are derived from sclerotomes. Ad-
vanced mineralization was found in the vertebrae throughout the
vertebral columns of Sox9?/?neonates (Fig. 5 E–J). The ossifi-
cation centers of the cervical vertebrae were larger or appeared
earlier than those of the corresponding vertebrae in wild-type
controls. In the thoracic regions, ossification in each dorsal-arch
unit already extended ventrally to fuse with the ossification
centers of the vertebral bodies (Fig. 5 G–J). Dissection of the
vertebral axis revealed that the shape of the vertebrae was
normal although occasionally part of the dorsal arch was missing
(Fig. 5H); cartilaginous elements in the dorsal parts of the
vertebrae stained more weakly with Alcian blue or were simply
missing (Fig. 5 F and H). Consistent with the premature min-
eralization of vertebrae in neonatal Sox9?/?mutants, sections
through equivalent caudal vertebrae of E15.5 embryos indicated
that the process of differentiation of hypertrophic chondrocytes
was advanced more in Sox9?/?mutants than in wild-type litter-
mates (Fig. 5 K and L).
The ossification centers of the talus and the calcaneum in the
hindlimbs were enlarged in the heterozygous mutants, and the
formed earlier in the heterozygotes also (data not shown).
Ossification occurred prematurely in both greater horns of the
hyoid bone, whereas it normally occurs only postnatally (Fig. 2
M and N).
We also analyzed the epiphyseal growth plates of the tibiae of
Sox9?/?neonates and E18.5 embryos by histological analysis and
were present in Sox9?/?mutants (Fig. 6 A and B); more
specifically, the zones of resting and proliferating chondrocytes
in Sox9?/?mutants appeared normal. Von Kossa staining
showed that the calcification of the terminal hypertrophic chon-
drocytes was normal (data not shown). However, histological
embryos at E12.5. (A and B) Whole embryos. (C and D) Forelimbs. (E and F)
Hematoxylin?eosin staining of transverse sections through equivalent tho-
racic regions. Cells in the sclerotome surrounding the notochord (arrowhead)
appeared like mesenchymal cells in the Sox9?/?mutants (F), but were typical
cobblestone-like prechondrocytic cells in wild-type controls (E). (G) Northern
analysis with total RNA from wild-type (???) and Sox9?/?mutant (???)
embryos at E10.5 and E12.5. The expression levels of Sox9 and Col2a1 were
percent expression levels of the mutant relative to wild type are shown. S,
scapula; H, humerus; R, radius; U, ulna; F, femur; Fi, fibula; T, tibia; Pv,
Alcian blue staining of wild-type (A and C) and Sox9?/?(B and D)
(A and B) Middle ears. (C and D) Dorsal view of the skull. (E and F) Seventh
cervical vertebrae. (G and H) Third thoracic vertebrae. (I and J) Von Kossa
staining of sections of fifth thoracic vertebrae. The cartilaginous regions
between the vertebral body and the pedicles of dorsal arches in wild-type
control (I) were mineralized in E18.5 Sox9?/?mutants (J). (K and L) Hematox-
ylin?eosin staining of sections of equivalent caudal vertebrae from E15.5
are indicated by arrowheads. Ip, interparietal bone; So, supraoccipital bone;
Vb, vertebral body; Da, dorsal arch; Id, intervertebral discs.
Premature mineralization in the Sox9?/?mutants. (A–H) Skeletal
Bi et al.
June 5, 2001 ?
vol. 98 ?
no. 12 ?
analysis of the growth plate at E18.5 showed that the hypertro-
phic zone in the Sox9 heterozygotes was enlarged (Fig. 6 A and
B). In situ hybridization indicated that type X collagen RNA was
expressed in a wider region in growth plates of Sox9?/?neonates
(Fig. 6 E and F). No major changes were seen in the pattern of
expression of parathyroid hormone receptor and of Indian
hedgehog (Fig. 6 G–J). The enlargement of the hypertrophic
zones of the epiphyseal growth plates in the absence of one
functional allele suggested that Sox9 regulates the rate of hy-
pertrophic chondrocyte differentiation.
Hypoplastic Cartilage Primordia Are Caused by Sox9 Haploinsuffi-
ciency. Heterozygous Sox9 mutant mice are characterized by
hypoplasia of all bones formed by endochondral ossification of
all cartilages. In E14.5 Sox9?/?mutant embryos, all cartilage
skeletal elements were already thinner or smaller. There were,
however, no obvious patterning defects, suggesting that Sox9
haploinsufficiency does not influence skeletal-pattern forma-
tion. The bending observed in several hypoplastic cartilaginous
bone precursors may be caused by fetal muscle contraction.
In E12.5 wild-type embryos, cartilages begin to form from
mesenchymal condensations, whereas in distal parts of limbs
mesenchymal condensations form from committed cells. At this
stage, all cartilages and cartilage primordia visualized by using
Alcian blue staining were hypoplastic. In addition, the chondro-
blasts surrounding the notochord in Sox9?/?embryos still had
the aspect of mesenchymal cells, whereas at equivalent locations
in wild-type embryos these cells were more chondrocytic. Con-
sistent with this finding, we found lower levels of Col2a1 RNA
in whole E12.5 and E10.5 Sox9?/?mutant embryos compared
with wild-type littermates. A reduction of Col2a1 RNA levels
was observed also by in situ hybridization of Col2a1 at E10.5 and
E11.5 (data not shown). The hypoplastic cartilages and cartilage
primordia in Sox9?/?mutants could be because of a reduced
proliferation rate of cells in mesenchymal condensations and in
forming cartilages. However, when cells present in E12.5 verte-
brae were labeled by BrdUrd or examined for proliferating cell
nuclear antigens, they showed the same density of proliferating
cells in Sox9?/?and wild-type embryos, indicating that haplo-
insufficiency of Sox9 did not affect the rate of cell proliferation
(data not shown). Given that Sox9 is required for chondrogenic
mesenchymal condensations, we propose that the hypoplastic
cartilages are caused by defective mesenchymal condensations in
heterozygous animals, perhaps because fewer cells are recruited
to the mesenchymal condensations. A study using simulations of
stochastic gene expression has suggested that inactivation of a
single allele of a gene in diploid organisms could decrease the
probability of expression of this gene, and that initiation of gene
expression could be delayed in the absence of one functional
needed for precartilaginous mesenchymal condensations are not
expressed at sufficient levels in the mutants. Correct expression
of these genes would need a threshold level of Sox9 that would
be reached less frequently in heterozygous mutants than in
wild-type embryos. It should be noted that the Kd for SOX9
binding to DNA is in the nanomolar range (19), in contrast to the
Kdof many other transcription factors that are in the picomolar
range. It is therefore conceivable that the absolute concentration
of SOX9 is near the Kdvalue in wild-type individuals, thus a 50%
reduction in Sox9 would affect transcription of at least some
Premature mineralization was observed in many skeletal ele-
ments of Sox9?/?mutant mice. Because expression of Sox9 RNA
and protein is shut off completely in the hypertrophic zone, this
premature mineralization is most likely a consequence of the
haploinsufficiency existing in Sox9-expressing cells that are
progenitors of the hypertrophic chondrocytes.
Despite the hypoplasia of cartilage primordia, the different
cellular components of the growth-plate cartilage were present
and their organization appeared normal. Thus the chondrocytes
were responding normally to signals that result in the proper
cellular organization of the growth plate. However, comparison
of the length of different zones in these growth plates showed
We propose that Sox9 has a role in regulating the rate of
chondrocyte differentiation into hypertrophy, and that Sox9
Table 1. Comparison of the skeletal abnormalities in patients
with CD and Sox9?/?mice
Small thoracic cage
Bowing of long bones
Bowed ulnae or radii
Bowed manubrium sternae
Deformed pelvis and spine
Missing pair of ribs
the proximal tibia. (A and B) Alcian blue staining of the epiphyseal growth
plates of wild-type (A) and Sox9?/?(B) embryos at E18.5. (C–J) RNA in situ
hybridization analysis of the expression of Col10a1 (E and F), parathyroid
tibia epiphyseal growth plates of wild-type (C, E, G, and I) and Sox9?/?(D, F,
H, and J) neonates. (C and D) Bright field. H, hypertrophic zone.
Enlarged hypertrophic zone in Sox9?/?epiphyseal growth plate of
www.pnas.org?cgi?doi?10.1073?pnas.111092198Bi et al.
might function in maintaining the chondrocyte phenotype by Download full-text
inhibiting hypertrophic chondrocyte differentiation. Sox9 hap-
loinsufficiency would result in accelerated hypertrophic differ-
entiation and subsequently in premature mineralization. The
normal inhibition of hypertrophic chondrocyte differentiation
may require a level of Sox9 activity that is not attained in the
prehypertrophic zone of the Sox9?/?mutants. This view is
supported by recent experiments suggesting that Sox9 might be
a physiological target of parathyroid hormone (PTHrP) signal-
ing, and might mediate at least in part the effects of PTHrP in
maintaining cells as nonhypertrophic cells (20). Altogether these
experiments favor the view that the function of Sox9 in chondro-
genesis extends beyond the step of mesenchymal condensations.
Analogies Between Skeletal Dysmorphologies of Sox9?/?Mice and
Patients with CD.ManyoftheskeletaldefectsobservedinSox9?/?
mice phenocopy those of the human disease CD as shown in
Table 1. Despite many striking similarities in the skeletal defects
of Sox9?/?mice and patients with CD, some differences exist
nonetheless. In patients with CD, bending of bones usually is
limbs (9, 10). In contrast, in Sox9?/?mutant mice, skeletal
elements in both forelimbs and hindlimbs, including ulnae, radii,
and tibiae, exhibited bending, whereas the proximal bones were
hypoplastic but did not show bending. These discrepancies could
be the result of relative skeletal differences in the overall shape
of specific cartilage primordia between humans and mice.
The skeletal defects of patients with CD show a wide variation
among affected individuals (10). In mice, the phenotypic man-
ifestations of the heterozygous Sox9 mutations were influenced
strongly by their genetic background. In Sox9?/?mutant mice on
the 129SvEv inbred background, the skeletal defects were more
severe. The genetic background effects might be caused by other
loci that encode proteins that cooperate with Sox9. Because
L-Sox5 and Sox6 have been shown to cooperate with Sox9 in
activating the Col2a1 and aggrecan (21), the expression of L-Sox5
and Sox6 could conceivably influence the effect of a given Sox9
Because several lines of evidence indicate that our targeted
mutation is a null allele (3), we can exclude the creation of a
dominant-negative-acting SOX9 mutant polypeptide. Thus, our
data demonstrate that the function of Sox9 is dosage-dependent,
and that the reduction in the expression level of Sox9 affects all
endochondral and cartilage skeletal elements.
In summary, analysis of Sox9?/?mutants indicated that two
steps in the pathway of chondrocyte differentiation are sensitive
to Sox9 dosage. Sox9 haploinsufficiency results in defective
cartilage primordia, which in turn are the causes for the hyp-
oplasia and the eventual bending of the cartilaginous and
endochondral skeletal elements. The premature mineralization
of many skeletal elements indicates that another step preceding
the formation of hypertrophic chondrocytes in the growth plate
is also sensitive to Sox9 dosage.
We thank Gerald Pinero and Heidi Ebspaecher for histological assis-
tance. We also thank R. Johnson for the Indian hedgehog probe and M.
Kronenberg for the parathyroid hormone receptor probe. This work was
funded by National Institutes of Health Grants P01 AR42919 (to B.deC.
and R.B.) and R01 HD30284 (to R.B.). DNA sequencing was supported
by National Institutes of Health Grant CA16672.
1. Tacchetti, C., Tracella, S., Dozin, B., Quarto, R., Robino, G. & Cancedda, R.
(1992) Exp. Cell Res. 200, 26–33.
2. Cancedda, R., Descalzi Cancedda, F. & Castagnola, P. (1995) Int. Rev. Cytol.
3. Bi, W., Deng, J. M., Zhang, Z., Behringer, R. R. & de Crombrugghe, B. (1999)
Nat. Genet. 22, 85–89.
4. Wright, E., Hargrave, M. R., Christiansen, J., Cooper, L., Kun, J., Evans, T.,
Gangadharan, U., Greenfield, A. & Koopman, P. (1995) Nat. Genet. 9, 15–20.
5. Zhao, Q., Eberspaecher, H., Lefebvre, V. & de Crombrugghe, B. (1997) Dev.
Dyn. 209, 377–386.
6. Wagner, T., Wirth, J., Meyer, J., Zabel, B., Held, M., Zimmer, J., Pasantes, J.,
Bricarelli, F. D., Keutel, J., Hustert, E., et al. (1994) Cell 79, 1111–1120.
7. Ng, L.-J., Wheatley, S., Muscat, G. E. O., Conway-Campbell, J., Bowles, J.,
Wright, E., Bell, D. M., Tam, P. P. L., Cheah, K. S. E. & Koopman, P. (1997)
Dev. Biol. 183, 108–121.
8. Foster, J. W., Dominguez-Steglich, M. A., Guioli, S., Kwok, C., Weller, P. A.,
Stevanovic, M., Weissenbach, J., Mansour, S., Yong, I. D., Goodfellow, P. N.,
et al. (1994) Nature (London) 372, 525–530.
9. Houston, C. S., Opitz, J. M., Spranger, J. W., MacPherson, R. I., Reed, M. H.,
Gilbert, E. F., Herrmann, J. & Schinzel, A. (1983) Am. J. Med. Genet. 15, 3–28.
10. Mansour, S., Hall, C. M., Pembrey, M. E. & Young, I. D. (1995) J. Med. Genet.
11. Mcleod, M. J. (1980) Teratology 22, 299–301.
12. Ojeda, J. L., Barbosa, E. & Bosque, P. G. (1970) Stain Technol. 45, 137–138.
13. Mertin, S., McDowall, S. G. & Harley, V. R. (1999) Nucleic Acids Res. 27,
14. Metsaranta, M., Toman, D., de Crombrugghe, B. & Vuorio, E. (1991) Biochim.
Biophys. Acta 1089, 241–243.
15. Coustry, F., Maity, S. N. & de Crombrugghe, B. (1995) J. Biol. Chem. 270,
16. Lefebvre, V., Huang, W., Harley, V. R., Goodfellow, P. N. & de Crombrugghe,
B. (1997) Mol. Cell. Biol. 17, 2336–2346.
17. Kaufman, M. H. & Bard, J. B. L. (1999) in The Anatomical Basis of Mouse
Development (Academic, London), pp. 58–59.
18. Cook, D. L., Gerber, A. N. & Tapscott, S. J. (1998) Proc. Natl. Acad. Sci. USA
19. Goldberg, H., Helaakoski, T., Garrett, L. A., Karsenty, G., Pellegrino, A.,
Lozano, G., Maity, S. N. & de Crombrugghe, B. (1992) J. Biol. Chem. 267,
20. Huang, W., Chung, U., Kronenberg, M. H. & Benoit de Crombrugghe. (2001)
Proc. Natl. Acad. Sci. USA 98, 160–165. (First Published December 19, 2000;
21. Lefebvre, V., Li, P. & de Crombrugghe, B. (1998) EMBO J. 17, 5718–5733.
Bi et al.
June 5, 2001 ?
vol. 98 ?
no. 12 ?