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DEVELOPMENTAL DYNAMICS 217:401– 414 (2000)
Positionally-Dependent Chondrogenesis Induced by BMP4
Is Co-Regulated by Sox9 and Msx2
ICHIRO SEMBA, KAZUAKI NONAKA, ICHIRO TAKAHASHI, KATSU TAKAHASHI, RALPH DASHNER,
LILLIAN SHUM, GLEN H. NUCKOLLS, AND HAROLD C. SLAVKIN*
Craniofacial Development Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases,
National Institutes of Health, Bethesda, Maryland
ABSTRACT
Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the
maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9,
and type II collagen demonstrate temporally and
spatially restrictive localization patterns suggestive
of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants
induced ectopic cartilage formation in the proximal
position of the explant. However, BMP4-soaked
beads implanted at the rostral position did not have
an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2
expression in the immediate vicinity of the BMP4
beads 24 hours after implantation. Control beads
had no effect on cartilage induction or Msx2 and
Sox9 expression. Sox9 was induced at all sites of
BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and
suggested that Msx2 expression was inhibitory to
chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery
strategy. Microinjection of the Msx2-adenovirus to
the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in
the abrogation of endogenous cartilage and the
down-regulation of type II collagen expression.
Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2.
Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear
to establish a threshold for Sox9 function and
thereby restricts the position of chondrogenesis. Dev
Dyn 2000;217:401–414. Published 2000 Wiley-Liss, Inc.†
Key words: BMP4; Sox9; Msx2; type II collagen;
Meckel’s cartilage; chondrogenesis;
†
Published 2000 WILEY-LISS, Inc.
This article is a US government
work and, as such, is in the public domain in the United States of America.
mouse embryo; mandibular process;
bead implantation; organ culture;
whole-mount in situ hybridization;
Alcian Blue staining; competitive
RT-PCR; adenovirus gene delivery
INTRODUCTION
Bone morphogenetic factor was used to describe the
induction of cartilage and bone formation when semipurified bone matrix was introduced into the subectodermal and myogenic tissues of the adult rat and rabbit
(Urist, 1965). Thereafter, these factors were characterized (Wozney et al., 1988) and revealed more than 15
related proteins belonging to the family of bone morphogenetic proteins (BMPs); which in turn belongs to
an even larger group of growth and differentiation factors, the transforming growth factor beta (TGF␤) superfamily (see recent reviews Reddi, 1997; Vortkamp,
1997; Urist, 1997; Wozney and Rosen, 1998). BMPs
signal through a system of type I and II transmembrane receptor serine/threonine kinases, which are
then modulated by cytoplasmic Smad proteins (see reviews Derynck et al., 1998; Kawabata et al., 1998). In
the adult, elevated expression of BMP4 has been associated with the pathological condition afflicting the
skeleton, fibrodysplasia ossificans progressiva (Shafritz et al., 1996; Kaplan and Shore, 1998; Lanchoney et
al., 1998). In the embryo, BMP2 and BMP4 have been
implicated to function during skeletogenesis (see review Hall and Miyake, 1995), as well as in tissues in
which epithelial-mesenchymal interactions mediate
morphogenesis and differentiation (Nuckolls et al.,
1998).
Despite these significant contributions, the molecular mechanisms of bone or cartilage induction by any
member of the family remain elusive, partly due to
limitations associated with using either in vivo systems
The first two authors contributed equally to this project and are
considered co-first authors.
Grant sponsor: National Institutes of Health; Grant number: Z01AR41114.
Ichiro Semba’s present address is Department of Oral Pathology,
Kagoshima University Dental School, Kagoshima, 890-8544, Japan.
*Correspondence to: Dr. Harold C. Slavkin, Craniofacial Development Section, NIAMS, NIH, 6 Center Drive, Building 6, Room 324,
MSC-2745, Bethesda, MD, 20892-2745.
Received 16 August 1999; Accepted 7 January 2000
402
SEMBA & NONAKA ET AL.
or serum-containing in vitro systems in which the interactive effects of many other growth and differentiative factors cannot be isolated from the direct actions of
BMP itself. Further, the functions of BMP2 or BMP4 in
the embryo cannot as yet be fully studied in genetically
engineered mouse models because targeted disruption
of either molecule or their type I receptor resulted in
early embryonic lethality rendering the role of BMPs
during organogenesis, including skeletogenesis uninformative (Mishina et al., 1995; Winnier et al., 1995;
Zhang and Bradley, 1996). Finally, BMPs are a diverse
group of proteins with pleiotrophic functions in multiple differentiation programs and developmental stages
(see recent reviews Hogan, 1996; Graff, 1997). The
precise microenvironmental milieu regulating a specific biological action of a specific BMP molecule is often
difficult to identify.
Therefore, in order to address what are the positional
and temporal controls required for BMP-induced skeletogenesis, and what specific molecular events are associated with the developmental process, we employed
a unique in vitro culture system in which embryonic
mouse mandibular processes were explanted under serum-free conditions and subjected to a variety of micromanipulations. We report that exogenous BMP4 delivered by a protein-soaked bead implantation method
induced ectopic cartilage formation, yet the extent of
chondrogenesis was dependent on the dose of BMP4
administered, the developmental stage of the embryonic tissue, and the position within the developing
mandible. These temporal and positional tissue responses were attributable to the induction and combinatorial expression of two transcription factors, Sox9
and Msx2. The combinatorial and antagonistic actions
of these molecules appeared to restrict ectopic cartilage
formation during mandibular morphogenesis.
RESULTS
Exogenous BMP4 Induced Ectopic Cartilage
Formation in Embryonic Mandibular Process
Cultured Under Serumless Conditions
Mouse embryos at embryonic day 10 (E10) (Fig. 1a),
corresponding to Theiler stage 18 with 40 – 44 somite
pairs, are at a stage of development during which the
anlagen for major organs are shaped and overt cytodifferentiation is initiated. The mandibular process of
the developing first branchial arch was isolated by
microdissection and explanted into an organ culture
system supported by serumless, chemically-defined
medium. The mandibular process (Fig. 1b) consisted of
bilateral prominences of epithelium enclosing ectomesenchymal cells, derivatives of cranial neural crest cells
that emigrated from the posterior midbrain and anterior segments of the hindbrain (rhombomeres 1 and 2)
(Lumsden et al., 1991; Sechrist et al., 1993; OsumiYamashita et al., 1994), and head mesoderm. The serumless and chemically-defined culture system facilitates the investigation of time- and positionaldependent autocrine and paracrine growth and
differentiation factors, without the numerous con-
Fig. 1. Embryonic mandibular process explant culture. Embryonic
day 10 (E10) mouse embryo (a) was microdissected to isolate the mandibular process (arrow) and placed in serumless, chemically-defined
explant culture. At the beginning of the culture period (b), the mandibular
process consisted of two halves fused at the midline. Gross morphogenesis and cytodifferentiation occurred by day 6 of culture (c). Meckel’s
cartilage was identified by Alcian blue staining (d). Protein-soaked beads
(arrowheads) were placed within the developing tissue to deliver exogenous proteins (b, c, d). Exogenous BMP4 delivered by this method
induced ectopic cartilage formation as revealed by Alcian blue staining
(d). Scale bar for (a) ⫽ 1mm, and for (b– d) ⫽ 150␮m.
Sox9 and Msx2 CO-REGULATE BMP4-INDUCED CHONDROGENESIS
Fig. 2. BMP4-soaked beads induced ectopic cartilage formation only
when placed at the proximal position of the developing mandibular process. 100ng/␮l BMP4 was delivered by bead implantation method into
the rostral (a, d) or proximal (b, e) positions and allowed to develop in
vitro for 6 days. Chondrogenesis was assayed by whole-mount Alcian
blue staining (a, b, c) and toluidine blue staining on histological sections
403
d, e, f). Only beads implanted into the proximal position induced ectopic
cartilage formation (b, e). PBS-soaked bead had no effect on chondrogenesis (c). The morphology of BMP4-induced cartilage (e) was similar to
endogenous Meckel’s cartilage (f); arrowheads indicating perichondrium.
All beads are circled. Rostral orientation is top of panel in (a– c). Scale bar
for (a, b, c) ⫽ 150␮m, and for (d, e, f) ⫽ 50␮m.
Fig. 3. BMP4, Msx2, and Sox9 exhibited restrictive expression patterns. Whole-mount in situ hybridization
for BMP4 (a– d), Msx2 (e– h) and Sox9 (i–l) on 0 (a, e, i), 2 (b, f, j), 4 (c, g, k), and 6 days (d, h, i) of culture.
Whole-mount Alcian blue staining demonstrated Meckel’s cartilage formation at 4 (m) and 6 days (n) of
culture. Rostral orientation is top for all panels. Scale bars for (a, e, i) and (b– d, f– h, j–n) ⫽ 100␮m.
404
SEMBA & NONAKA ET AL.
TABLE 1. Frequency of Ectopic Cartilage Induced by
BMP4-Soaked Beads in Mandibular Processes in
Explant Cultures
Concentration of
BMP4 (ng/␮l)
Rostral
Proximal
10
0/15 (0%)
0/15 (0%)
50
0/15 (0%)
6/15 (40%)
100
0/18 (0%)
15/18 (83.3%)
founding variables found in serum supplement and the
maternal circulation. Growth and differentiation of the
mandibular process in vitro are delayed but not retarded when compared with in vivo development
(Slavkin et al., 1989). Further, the explant system supports micromanipulations such as bead implantation
and the microinjections used in this study.
Beads soaked in BMP4 were positioned efficiently
and reproducibly in the mandibular process at the beginning of culture (Fig. 1b) and the biological effects
observed over a period of six days. By six days in
culture (Slavkin et al., 1999; Fig. 1c), differentiation
and the formation of cartilage, osteoid, incisor and molar tooth buds and tongue were observed. Whole-mount
Alcian blue staining demonstrated the formation of
Meckel’s cartilage which was composed of a rostral
triangular-shaped piece, bilateral rod-shaped pieces
and proximal pieces representing the future middle ear
ossicles (Fig. 1d). Beads soaked in 100ng/␮l BMP4 consistently induced ectopic cartilage formation (Fig. 1d).
Ectopic cartilage was observed to fuse with endogenous
cartilage formation patterns resulting in expansion or
bifurcation morphology, or as observed as isolated ectopic cartilage pieces.
Ectopic Chondrogenesis Induced by BMP4 Was
Positional-, Temporal-, and Dose-Dependent
Further experiments demonstrated that beads
soaked in 100ng/␮l BMP4 only induced ectopic cartilage formation when the bead was placed in the proximal position of the developing mandibular process
when compared with the rostral position (Fig. 2a, b;
Table 1). Control beads soaked in PBS (Fig. 2c), or
beads soaked in 100ng/␮l FGF8 (data not shown), did
not induce ectopic chondrogenesis or disrupt endogenous cartilage formation. Because Alcian blue staining
demonstrated positive reaction to sulfated proteoglycan suggestive of cartilage, we additionally performed
routine histological evaluation on frozen sections of the
developing mandiblular explants at the site of bead
implantation. Consistent with the whole-mount Alcian
blue staining results, cartilage was observed enveloping beads implanted at the proximal position (Fig. 2e),
whereas cartilage was not detectable surrounding
beads implanted at the rostral position (Fig. 2d). Cartilage induced by BMP4 has morphological and histological characteristics similar to that of normal cartilage (Fig. 2f), including the presence of an intact
perichondrium and mature chondrocytes residing
within lacunae of the cartilagenous matrix. No signif-
icant apoptosis was detected associated with bead implantation.
The incidence of ectopic chondrogenesis was directly
dependent on the concentration of BMP4 used in these
investigations (Table 1). At 10ng/␮l BMP4, no ectopic
cartilage was induced, whereas at 100ng/␮l the incidence was 83.3% when beads were implanted at the
proximal position.
BMP4 Induced Different Levels of Sox9 and
Msx2 Expression Dependent on Positional
Information
To explain positional dependency of BMP4 induced
chondrogenesis, whole-mount in situ hybridizations,
using several gene probes at various time points, was
performed to identify candidate molecules associated
with the phenomenon. Mandibular processes at the
beginning of culture, and after 2, 4, or 6 days of development were fixed and processed. Endogenous BMP4
transcripts were detected in the E10 mandibular process, with a higher level of expression on the medial
aspect when compared with the lateral (Fig. 3a); consistent with previous reports (Bennett et al., 1995;
Neubüser et al., 1997). Thereafter, BMP4 exhibited
complex localization patterns (Figure 3b– d), presumably due to its functions in multiple cell lineages within
the developing mandibular process— chondrogenic and
odontogenic. The localization pattern of Msx2 in the
mandibular process in culture in part overlapped with
that of BMP4 expression especially in the rostral area,
yet was more restrictive (Fig. 3e– h). At day 2 of culture, Msx2 was expressed in rostral-medial and bilaterally symmetrical proximal-lateral sites, which appeared to delineate the rostral and caudal extent of
future Meckel’s cartilage formation (Fig. 3f ). Further,
at day 4 of culture Msx2 expression was observed along
a boundary interface with sites destined to become
cartilage (Fig. 3g).
Sox9 has been shown to be a direct transcriptional
activator of the cartilage-specific type II collagen gene
(Lefebvre et al., 1997). The localization of endogenous
Sox9 in the developing mandibular process was associated with Meckel’s cartilage development (Figure 3i–l).
At day 2 of culture, prior to cartilage differentiation,
Sox9 was localized in a rostral triangular-shaped site,
bilateral sigmoidal-shaped sites and at the proximal
lateral margins of the explants (Fig. 3j). This pattern
resembled the Alcian blue staining pattern of Meckel’s
cartilage at day 6 of culture (Fig. 3n), and presumably
served as a morphogenetic template for this cartilage.
Sox9 expression was somewhat decreased but still detectable at day 4 and 6 of culture (Fig. 3k, l) at which times
cartilage differentiation was observed (Fig. 3m, n).
Because the endogenous expression pattern of Msx2
overlapped with that of BMP4, and that it also appeared to define boundary interfaces suggestive of a
patterning role during chondrogenesis, the expression
of Msx2 in mandibular processes implanted with
BMP4-soaked beads was examined after 24 hours of
culture (Fig. 4a, b). Consistent with the responses of
Sox9 and Msx2 CO-REGULATE BMP4-INDUCED CHONDROGENESIS
many other developmental stages and tissues, BMP4
induced Msx2 expression in the immediate vicinity of
the source. However, the level of induced Msx2 expression appeared to be higher when the BMP4-soaked
bead was implanted in the rostral as compared with
that in the proximal position. This expression of Msx2
in response to BMP4 was transient and down-regulated in the following 24 hours (Fig. 4c, d).
Because Sox9 is a direct transcription activator of
the type II collagen gene, we examined its expression in
response to exogenous BMP4. One day after bead implantation, Sox9 expression was induced surrounding
the BMP4-soaked bead (Fig. 4e, f ). Contrary to Msx2
induced expression, the amount of Sox9 was higher
around the BMP4-soaked bead implanted at the proximal as compared with the rostral positions. Similar to
the Msx2 response, induced Sox9 expression was decreased by day 2 of explant culture (Fig. 4g, h).
The expression of type II collagen, presumably mediated by the activation of Sox9, was detected surrounding the BMP4-soaked bead at 48 hours of culture;
when Sox9 expression was already down-regulated
(Fig. 4j). This observation is consistent with previous
reports of Sox9 expression preceding that of type II
collagen (Ng et al., 1997; Takahashi et al., 1998a). The
expression of type II collagen at 24 and 48 hours
showed a pattern that was predictable of the differentiated phenotype as detected by Alcian blue staining on
day 4. Type II collagen expression was detected around
the developing Meckel’s cartilage. In addition, expression was also detected around the BMP4-soaked bead
implanted at the proximal position, resulting in an
expansion of the domain (Fig. 4j, l). Type II collagen
expression, though detected surrounding the BMP4soaked bead implanted at the rostral position at 24
hours of culture (Fig. 4i), was observed to be downregulated to non-existence by 48 hours (Fig. 4k). This
change in expression level was repeatedly consistent
with the phenotypic outcome at day 6, when Alcian
blue staining did not detect any ectopic chondrogenesis
when BMP4-soaked beads were introduced at this position.
The molecular responsiveness of the tissue to BMP4
was restricted to the first 24 hours of culture. BMP4soaked beads implanted into the mandibular process
after the explants had been in culture for one day
showed no induction of Msx2 nor Sox9, and no ectopic
cartilage (data not shown). Taken together, these data
demonstrate that tissue response to exogenous BMP4
is positionally and temporally dependent.
The observed difference in the expression of BMP4
inducible genes was further analyzed using quantitated morphometric (Table 2) and semi-quantitative
RT-PCR approaches (Fig. 5). First, to minimize variations in developmental stage and thereby responsiveness among different E10 explants, two beads were
implanted into the same piece of tissue; one in each of
the rostral and proximal positions, respectively. Consistent with the single bead implantation experiments,
the rostral position demonstrated a more intense re-
405
sponse to Msx2 (Fig. 4m), and a weaker signal for Sox9
(Fig. 4n), when compared with the proximal position.
Type II collagen expression was detected at both sites
of implantation (Fig. 4o). Control beads soaked in PBS
had no effect on gene expression level or pattern of
either Msx2 (Fig. 4p), Sox9 (Fig. 4q) or type II collagen
(Fig. 4r). Subsequently, explants processed for wholemount in situ hybridization were subjected to morphometric analyses in which the area of induced gene
expression was converted to the number of pixels positive for the hybridization signal (Table 2). With over
nine explants analyzed for each gene pattern, the
quantitative analysis was consistent with the qualitative analysis. Expression of Sox9 surrounding the
BMP4-bead implanted at the proximal position was 2.6
times higher than that at the rostral position. In contrast, Msx2 expression was three-fold weaker, comparing the corresponding positions. Expression of type II
collagen was 6.1-fold higher surrounding the proximally placed BMP4-beads than those placed rostrally.
The differences in size of induced Sox9, Msx2, and type
II collagen expression between the rostral and proximal positions were statistically significant (P ⬍ 0.01).
Semi-quantitative RT-PCR results also supported
positionally-dependent differential levels of induction
of Sox9 and Msx2 by BMP4. Consistent with data from
both whole-mount in situ hybridization and morphometric analyses, a three-fold elevation of Sox9 induced
by BMP4 was detectable when beads were implanted
proximally as compared with beads implanted rostrally, or controls using PBS-soaked beads or no beads
(Fig. 5a). We could not detect a significant increase in
Sox9 expression induced by BMP4-soaked beads placed
at the rostral position, possibly due to the high endogenous level of Sox9 expression marking the morphogenetic template of Meckel’s cartilage. Significant induction of Msx2 expression by BMP4 was also detected at
both rostrally or proximally implanted beads; more
than three-fold and almost two-fold, respectively, when
compared with controls (Fig. 5b). The difference between rostral and proximal expression levels was statistically significant (P ⬍ 0.05).
Direct Over-Expression of Msx2 Inhibited
Chondrogenesis
Sox9 expression preceded that of type II collagen, yet
type II collagen expression was not maintained around
the BMP4-soaked bead implanted at the rostral position, resulting in no ectopic cartilage. This could be due
to the expression of Msx2, which was much more
heavily detected at the rostral rather than the proximal position. To test the hypothesis that Msx2 inhibits
BMP4-induced chondrogenesis, a recombinant adenovirus was engineered to over-express the Msx2 gene
(AdV-Msx2). The adenovirus was microinjected into
the developing mandibular process and its actions evaluated against explants microinjected with control adenovirus bearing LacZ expression (AdV-LacZ). Consistent with previous reports (Takahashi et al., 1998b),
microinjection of AdV-LacZ resulted in high levels of
406
SEMBA & NONAKA ET AL.
Fig. 4. BMP4 induced Sox9 and Msx2 expression in developing
mandibular process in explant cultures. Beads soaked in 100ng/␮l BMP4
were implanted into the rostral (a, c, e, g, i, k) or proximal (b, d, f, h, j, l)
positions of mandibular processes and explanted into cultures. Wholemount in situ hybridization was performed for Msx2 (a– d), Sox9 (e– h),
and type II collagen (i–l), after 24 (a, b, e, f, i, j), or 48 (c, d, g, h, k, l) hours
in culture. The induced expression of Msx2 and Sox9 was detected after
24 hours, whereas the inductive effect was down-regulated after 48 hours
in culture. Sox9 expression appeared higher surrounding the BMP4soaked bead placed at the proximal (f) than that at the rostral (e) position,
whereas this response appeared to be reverse for that of Msx2. Type II
collagen was only observed surrounding BMP4-soaked beads implanted
at the proximal postition (j). In order to demonstrate that positionaldependent differential gene induction was not due to differences among
individual mandibles, multiple BMP4-soaked beads were implanted at
various positions within the same mandible and whole-mount in situ
hybridization was performed for Msx2 (m), Sox9 (n) and type II collagen
(o) after 1 day in culture. The results were similar to that of the single
bead implantation experiments. Control PBS-soaked beads implanted at
any position did not induce Msx2 (p), Sox9 (q) or type II collagen (r) after
24 hours in culture. All beads are circled. Rostral orientation is top for all
panels. Scale bars ⫽ 100␮m.
expression detectable 36 hours subsequent to injection
(Fig. 6a). AdV-LacZ did not affect BMP4-induced ectopic cartilage formation in the proximal position of the
mandibular process (Fig. 6b). When AdV-Msx2 was
microinjected at the same site where BMP4-soaked
bead was implanted, ectopic cartilage formation was
reduced (Fig. 6c, d) or abolished (Fig. 6e). The cartilage
matrix was weakly stained with Alcian blue and appeared vacuolated.
In addition, we also tested whether over-expression
of Msx2 inhibits endogenous cartilage formation. The
recombinant adenovirus was injected into the medial
or lateral side of the developing mandibular process.
Similarly, high expression of lacZ was detectable at 36
hours subsequent to injection (Fig. 7a, b). AdV-LacZ
injected explants had no deleterious effects and Meckel’s cartilage developed normally (Fig. 7c, d). In contrast, AdV-Msx2 injected into the lateral side of the
mandibular process resulted in an abrogation of Meckel’s cartilage formation, whereas neither AdV-Msx2 injected into the medial part of the explant, nor the
uninjected side produced Meckel’s cartilage dysmorphogenesis. Histological analyses confirmed that normal cartilage formed in the control non-injected side
(Fig. 7g), yet no evidence of cartilage was observed at
the AdV-Msx2 injected side of the explant (Fig. 7h). No
significant apoptosis was detected in adenovirus injected specimens.
Sox9 and Msx2 CO-REGULATE BMP4-INDUCED CHONDROGENESIS
407
TABLE 2. Size of Sox9, Msx2, and Type II Collagen Expression Domain Induced by 100 ng/␮l BMP4-Soaked Beads
in Mandibular Processes in Explant Cultures
Sox9
Msx2
Type II Collagen
Rostral mean ⫾ SD (pixels)
919.8 ⫾ 460.2
2809.2 ⫾ 632.6
257.3 ⫾ 75.3
Proximal mean ⫾ SD (pixels)
2346.0 ⫾ 578.1*
890.1 ⫾ 207.3*
1572.2 ⫾ 463.4*
Proximal/rostral ratio
2.55
0.32
6.11
*P ⬍ 0.01.
The level of type II collagen expression subsequent to
AdV-Msx2 microinjection was also analyzed by competitive PCR technique in explants after 6 days in culture
(Fig. 7i). Consistent with the morphological analyses,
there was no difference in type II collagen expression
when Msx2 was locally infected in the medial aspect of
the developing mandibular process. In contrast, when
Msx2 was over-expressed in the lateral side of the
mandible, the amount of type II collagen expression
was reduced to only 5% of control value (p ⬍ 0.001).
DISCUSSION
The induction of bone and cartilage by BMPs has
been well documented in both the embryo and the
adult. In the embryo, endogenous actions of BMPs contribute to skeletogenesis (Lyons et al., 1989; FrancisWest et al., 1994; Dewulf et al., 1995; Reddi, 1994,
1995; Hall and Miyake, 1995; Frenz et al., 1996;
Kawakami et al., 1996; Zou et al., 1997) and exogenous
applications result in ectopic bone and cartilage formation (Duprez et al., 1996a; Francis-West et al., 1996;
Barlow and Francis-West, 1997; Ekanayake and Hall,
1997; Macias et al., 1997; Nifuji et al., 1997). Consistent with the literature, we first confirmed that such
osteo-chondro-inductive potential of BMP4 also functions in the embryonic mandibular mesenchyme (Fig.
1). We extended these observations to the transcription
level, and identified and characterized the mechanism
of BMP4 action using a serumless, chemically-defined
explant culture system model (Figs. 1 and 2). We observed that ectopic cartilage formation induced by
BMP4 was preceded by the up-regulation of Sox9 and
Msx2 (Fig. 4). However, overt chondrogenesis was only
defined by the relative high expression level of Sox9
when compared with that of Msx2 (Figs. 4 and 5). In
contrast, elevated levels of Msx2 inhibited ectopic cartilage induction by BMP4 despite the concomitant expression of Sox9. Because Sox9 has been shown to be a
direct activator of the type II collagen promoter, we
hypothesize that Msx2 is a repressor of chondrogenesis. Indeed, over-expression of Msx2 by adenoviral gene
delivery in the mandibular explants inhibited BMP4induced chondrogenesis (Fig. 6) and also resulted in
the down-regulation of type II collagen expression and
abrogation of Meckel’s cartilage formation (Fig. 7). The
combinatorial actions of these two transcription factors
regulate the extent of chondrogenesis and appear to
define the temporal and positional information of tissue responses to BMP4.
Fig. 5. BMP4 induced different levels of Sox9 and Msx2 expression
according to positional information in developing mandibular process in
explant cultures. Relative expression of Sox9 (a) or Msx2 (b) was assayed by semi-quantitative RT-PCR on mandibular processes implanted
with 100ng/␮l BMP4-soaked beads at the rostral or proximal position, and
then compared with controls which consisted of non-implanted or PBSsoaked bead implanted specimens. *P ⬍ 0.05 compared with control,
✣ P ⬍ 0.05 compared with proximal value. N ⫽ 8 or 9 for all groups.
408
SEMBA & NONAKA ET AL.
Fig. 6. Over-expression of Msx2 inhibited BMP4induced chondrogenesis. Adenovirus expressing LacZ
(a, b) or Msx2 (c, d, e) was microinjected at the same
site where BMP4-soaked beads were implanted at the
rostral position of the mandibular process. Beta-galactosidase staining of mandibular processes after 36
hours in culture confirmed the site and spread of lacZ
expression (a). Whole-mount Alcian blue staining of
infected mandibular processes after 6 days demonstrated that ectopic chondrogenesis induced by BMP4
was unaffected by lacZ expression (b) but was reduced (c, d) or abolished (e) by Msx2 expression. The
affected cartilage appeared vacuolated and weakly
stained with Alcian blue (arrows). Scale bar ⫽ 100 ␮m.
Combinatorial Signaling Contributes to the
Diverse Functions of BMPs
Due to the pleiotropic nature of BMPs, an in vitro
serum-free culture system is invaluable for studies designed to identify and isolate the direct actions of BMP
without confounding complications from interacting serum factors. Previous attempts in using serum-free
systems to study chondrocyte maturation have provided initial phenotypic and biochemical evidence to
BMP-induced chondrogenesis, but the molecular basis
has yet to be elucidated (Leboy et al., 1997; Lee and
Chuong, 1997). Our studies have confirmed BMP-induced chondrogenesis in serumless conditions. In addition, we identified the early molecular responses of
cartilage initiation and demonstrated that Sox9, a
marker of chondrogenesis, is induced by BMP4. Because BMP can also induce the expression of Cbfa1
(Ducy et al., 1997), marker for osteogenesis, it is likely
that either the two skeletal cell lineages are segregated
at an early time point of differentiation or that the
action of BMP is temporally and positionally dependent.
It is conceivable that biological signaling molecules
interact synergistically or antagonistically with other
molecules to specify the temporal and spatial differentiation program (Davidson, 1995). BMP4 exemplifies
this concept due to its particularly diverse set of functions during embryogenesis. The action of BMP4 can be
regulated at multiple hierarchical levels to achieve cell
and tissue type specificity. At the ligand level, BMPs
act antagonistically with FGFs to regulate Pax9 ex-
pression (Neubüser et al., 1997), to specify tooth identity (Tucker et al., 1998), and to regulate limb outgrowth (Niswander and Martin, 1993; Buckland et al.,
1998). BMP also antagonizes with Shh to regulate cartilage formation in the vertebrae (Watanabe et al.,
1998). At the level of cytoplasmic signaling, BMP competes with EGF or HGF for activating versus inhibiting
phosphorylation signal on Smad1 (Kretzschmar et al.,
1997; Nonaka et al., 2000). Our results suggest that at
the transcription level, BMP4 induces two antagonistic
signals, which in turn regulate the chondrogenic pathway. This combinatorial regulatory sequence would appear to ensure the fidelity of responses elicited by a
single extracellular signal.
Threshold Level Exists for Sox9-Directed
Chondrogenesis
The Sry-type HMG-box containing transcription factor Sox9 is a direct transcriptional activator of the type
II collagen promoter (Bell et al., 1997; Lefebvre et al.,
1997); a signaling factor for chondrogenesis. Mutations
in Sox9 gene in human are associated with the skeletal
dysmorphology syndrome, campomelic dysplasia (Foster et al., 1994; Wagner et al., 1994). Expression of
Sox9 is associated with chondrogenesis (Wright et al.,
1995; Healy et al., 1996; Wheatley et al., 1996; Ng et
al., 1997; Zhao et al., 1997). Misexpression of Sox9
results in ectopic cartilage formation (Kanzler et al.,
1998; Healy et al., 1999) whereas teratomas with inactivated Sox9 contained no cartilage (Bi et al., 1999)
suggesting that Sox9 is sufficient to induce or promote
Sox9 and Msx2 CO-REGULATE BMP4-INDUCED CHONDROGENESIS
409
Fig. 7. Over-expression of Msx2 in developing mandibular processes
inhibited Meckel’s cartilage formation. Adenovirus-expressing LacZ (a– d)
or Msx2 (e, f) was microinjected to the medial (a, c, e) or lateral portions
(b, d, f) of the right half of the E10 mandibular process as shown in the
figure. Beta-galactosidase staining of mandibular processes after 36
hours in culture confirmed restrictive gene expression delivered by adenovirus microinjection and infection method (a, b). Whole-mount Alcian
blue staining of infected mandibular processes after 6 days demonstrated
no effect on Meckel’s cartilage formation by LacZ expression (c, d) or
Msx2 expression to the medial portion (e). Expression of Msx2 at the
lateral portion of the mandibular process inhibited Meckel’s cartilage
formation (f). Toluidine blue staining of histological sections after 6 days
revealed Meckel’s cartilage (outlined in white) on the uninjected side (g)
but absence of chondrogenesis or excessive apoptosis on the Msx2
expressing side (h). Scale bars for (a–f) ⫽ 100␮m, and for (g, h) ⫽ 50␮m.
Expression level of type II collagen was assayed by quantitative RT-PCR
in mandibular processes infected with adenovirus expressing LacZ
(hatched bars) or Msx2 (solid bars) at the medial or lateral portion (i).
Consistent with morphological analyses, Msx2 expression at the lateral
portion of the mandibular process inhibited type II collagen expression.
*P ⬍ 0.001. N ⫽ 6.
chondrogenesis. It has also been reported that Sox9
may bind and activate the promoters of two other cartilage markers, aggrecan and type X collagen (Harada
et al., 1996; Sekiya et al., 1997). Despite evidence supporting the critical role of Sox9 during early chondrogenesis, further upstream regulatory elements of this
pathway have been elusive.
It has been implicated that the level of Sox9 expression is significant in determining whether chondrogenesis is to occur or not. High levels of Sox9 are
observed to correlate with all sites of cartilage formation. Low levels of Sox9 are detected in a broad
array of non-chondrogenic embryonic tissues such as
notochord, neural tube, heart and lung (Ng et al.,
1997; Zhao et al., 1997). It is yet unclear whether an
absolute threshold level of Sox9 expression is required to induce chondrogenesis, nor how this level is
determined and controlled. Our data suggests that a
relative threshold level may exist. The expression of
Sox9 precedes that of type II collagen expression, an
indication that the responding mesenchymal cells
can and have initiated the chondrogenic program
regardless of the absolute expression level of Sox9.
However, overt chondrogenesis only occurs at sites
where Sox9 expression is high relative to that of the
repressor Msx2. Therefore, the antagonism between
Sox9 and Msx2 appears to establish a relative
threshold for Sox9 function in cartilage induction,
and that this relationship is site specific. This
threshold may also be modulated positively or nega-
410
SEMBA & NONAKA ET AL.
tively by the presence of other transcription factors
such as other Sox proteins (Lefebvre et al., 1998).
Msx2 Is a Repressor of Chondrogenesis
The Msx family of transcription factors is multifunctional, although very little is known of Msx3. Msx1 and
Msx2 are largely co-expressed in the developing embryo, and are thought to be redundant in functions
(Davidson, 1995; Maas et al., 1996). Disruption of Msx
functions results in multiple malformations including
that of the craniofacial skeleton (Satokata and Maas,
1994; Liu et al., 1995; Foerst-Potts and Sadler, 1997;
Winograd et al., 1997). In many cases, Msx genes mediate the function of BMPs including cranial suture
morphogenesis (Kim et al., 1998), apoptosis (Marazzi et
al., 1997; Takahashi et al., 1998b), tooth formation
(Tureckova et al., 1995; Jernvall et al., 1998), dorsalization of midline structures along the neural tube
(Takahashi et al., 1996), vertebral development (Monsoro-Burq et al., 1996; Watanabe and Le Douarin,
1996), and mammary gland development (Phippard et
al., 1996). Further, Msx1 and Msx2 are considered to
be general repressors of transcription (Catron et al.,
1995; Semenza et al., 1995), directly modulating the
function of the promoter for osteocalcin (Newberry et
al., 1997), type I collagen (Dodig et al., 1996), and
MyoD enhancer (Woloshin et al., 1995).
Consistent with a repressor function of Msx, our
investigations have provided direct evidence that expression of Msx2 is inhibitory in the chondrogenic
pathway; including both endogenous as well as ectopic
cartilage formation. This is supported by previous reports in which application of antisense oligonucleotides
directed against Msx2 resulted in an increase in cartilage formation in the avian mandible (Mina et al.,
1996). The repressor action of Msx2 is likely to be
complex. For example, it is unclear how Msx2 overexpression eliminated endogenous cartilage from the
lateral side of the mandibular process and not the
medial side. We speculate that because the medial
portion is a site of high endogenous expression of Msx1
and Msx2, our adenovirus-driven ectopic expression
might not have achieved an additionally increased
level. The endogenous ratio of Sox9 to Msx2 would then
already determine the outcome of chondrogenesis. It is
also possible that chondroprogenitor cells at the medial
position are less committed in phenotype and less responsive to Msx2 than those at the lateral. The lateral
part of Meckel’s cartilage was formed by day 4 of culture (Fig. 4m), whereas the medial portion was Alcian
blue positive only by day 6 (Fig. 4n). Because adenovirus expression in our system is maintained for 48 –72
hours, there is the potential for compensatory growth
when expression level declines. In addition, other results indicate that Msx1 or Msx2 can interact with
other transcription factors and compete for DNA binding (Catron et al., 1995; Zhang et al., 1996; Wu et al.,
1997; Zhang et al., 1997; Bendall et al., 1998). These
additional regulatory networks may be different locally. Taken together, these results suggest that the
regulation of a single extracellular signaling molecule
by combinations of transcription factors would appear
to be required to achieve a specific response in a specific
developmental context.
The Developing Mandibular Process Is
Compartmentalized
Compartmentalization of the developing mandibular
mesenchyme is associated with early complex molecular patterning. The cranial to caudal and proximal to
rostral axial plans are established and maintained by
the expression of a sequence of homeobox-containing
genes. The oral to aboral axis along the maxillary and
mandibular processes of the first branchial arch is associated with the expression of Lim-domain homeoproteins, Lhx6 and Lhx7 (Grigoriou et al., 1998). The oral
mesenchyme is thus competent to respond to epithelial
odontogenic signals such as FGFs. These axes are further refined by homeobox containing transcription factors of the Msx and Dlx families, and may specify the
formation of incisor versus molar teeth within the first
branchial arch (Qiu et al., 1997; Thomas et al., 1997).
Other domains have been suggested to limit the manifestation of other phenotypes, such as bone, cartilage
and muscle within the embryonic craniofacial mesenchyme. We propose that further compartmentalization
along the rostral-proximal and medial-lateral axes exists that are defined by the chondrogenic potential in
the forming mandible. This assertion is supported by
experiments in which meticulous microdissections followed by grafting, tissue recombination or micromass
cultures of different segments of the developing mandible, identified varying potentials to form cartilage
(Hall, 1982; Langille, 1994). Similar positional information also exists in the developing limb bud and has
been defined by the proximodistal, dorsoventral, and
anteroposterior axes (Cottrill et al., 1987; Ros et al.,
1997). Although not assayed in this study, it is likely
that BMP2, BMP7, and Msx1 may also be involved in
developing mandibular cartilage based on their sequence homologies and overlapping expression pattern
with BMP4 and Msx2, respectively. The specificity of
these families of growth and transcription factors towards chondrogenesis versus odontogenesis remains to
be characterized.
During early development, progressively restrictive
molecular patterning emerges from a sequence of transcriptional activations and inactivations. Our results
suggest that within the presumptive myogenic region
destined to be the tongue, chondrogenic potential exists
and that mesenchymal cells can be induced to become
chondrocytes in response to BMP4. The induction is
dose-dependent on exogenous BMP4, and suggests that
the endogenous developmental program consists of silencers to the chondrogenic pathway in the tuberculum
impar. Indeed, the induction of cartilage by BMP may
be the result of simultaneous regulation of two pathways; stimulation of chondrogenesis and inhibition of
myogenesis. In chick limb bud micromass cultures,
BMP2 specifically promotes chondrogenesis presum-
Sox9 and Msx2 CO-REGULATE BMP4-INDUCED CHONDROGENESIS
ably at the expense of myogenesis (Duprez et al.,
1996b). In the developing somite, BMP signaling inhibits the activation of MyoD and Myf5 (Reshef et al.,
1998). Further, in vitro studies support that endogenous Msx1 can bind to and repress the MyoD enhancer
(Woloshin et al, 1995). These findings and our results
indicate that BMP is instructive to both chondrogenesis as well as myogenesis in undifferentiated mesenchymal cells.
CONCLUSION
In summary, the evidence supports the concept that
the emergence of the chondrogenic potential of BMP4
from the pleiotropic nature of this growth factor is
mediated by the induced expression of Sox9 and Msx2.
Sox9 and Msx2 function as positive and negative regulators of chondrogenesis, respectively. The combinatorial action of these transcription factors appears to
define the extent of embryonic mouse mandibular
chondrogenesis.
EXPERIMENTAL PROCEDURES
Mouse Embryo and Mandibular Process
Explant Culture
Timed pregnant Swiss Webster mice were obtained
(Harlan Bioproducts for Science, Indianapolis, IN) and
embryos were collected at embryonic day 10 (E10) or
E12. Isolated embryos were further staged by external
morphology and number of somite pairs according to
Theiler staging system (Theiler, 1989). Organ culture
of mandibular processes from E10 embryos was performed according to previously reported protocols
(Shum et al., 1993; Slavkin et al., 1999). Briefly, the
mandibular processes were isolated by microdissection
and explanted into a Trowell-type organ culture system
using serumless, chemically-defined BGJb medium
(Life Technologies Inc., Gaithersburg, MA) supplemented with 100 ␮g/ml ascorbic acid and 100 U each of
penicillin and streptomycin, and allowed to develop at
37°C and 5% CO2.
Bead Implantation
Affi-Gel blue agarose beads (Bio-Rad Labs., Hercules, CA) at 100 –200 mesh, which correspond to a
diameter of 50 –75 ␮m were selected, washed twice in
phosphate buffered saline (PBS), and soaked in 10, 50,
or 100 ng/␮l human recombinant BMP4 (Genetics Institute Inc., Cambridge, MA), or PBS as control for 1 hr
at room temperature. BMP4-soaked or PBS control
beads were positioned by fine forceps and implanted
into the mandibular process explants using a mouthcontrolled micropipette under the stereomicroscope
(Slavkin., et al. 1999).
Whole-Mount Alcian Blue Staining
The presence of sulfated proteoglycans indicative of
cartilage formation was detected by Alcian Blue staining as previously described (Shum et al., 1993), using
0.04% Alcian Blue 8GX (Sigma, St. Louis, MO) in acid
411
ethanol followed by tissue clearing with a graded series
of potassium hydroxide and glycerol.
Histology
Specimens were fixed in 4% paraformaldehyde in 0.1
M PBS (pH 7.4) for 12 hr at 4°C. Subsequently, specimens were rinsed thoroughly in 0.01 M PBS, infiltrated
through a graded series of sucrose up to 30% and finally in 10% acrylamide and 30% sucrose in 0.01 M
PBS, embedded first in 10% acrylamide and then in
Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC). Seven micron-thick frozen sections were collected and stained with toluidine blue.
Whole-Mount In Situ Hybridization
Probes for BMP4 (GenBank X56848: 776-1248nt),
Sox9 (Wright et al., 1995, 926-1683nt), and type II
collagen (Metsaranta et al., 1991, GenBank M65161:
29648-31343nt) were obtained by RT-PCR method and
confirmed by direct sequencing. Probe for Msx2 was an
AluI to EcoRI fragment of the full length Msx2 cDNA
(Bell et al., 1993, GenBank L11739: 251-708nt). Digoxigenin (DIG)-labeled sense and antisense riboprobes for
mouse BMP4, Sox9, Msx2, and type II collagen were
prepared by in vitro transcription of linearized pBlueScript (Stratagene, La Jolla, CA) or pCRII (Invitrogen
Corp., Carlsbad, CA) phagemids containing cDNA insert using RNA Transcription Kit (Stratagene, La
Jolla, CA) according to specifications from the manufacturer.
Embryos and mandibular process explants were
fixed overnight in 4% paraformaldehyde in PBS at 4°C,
and processed according to the protocol described by
Rosen and Beddington (1993) with minor modifications. Briefly, after fixation, the specimens were
bleached for 1 hr with 6% hydrogen peroxide in PBS
containing 0.1% Tween-20 at room temperature. Mandibular explants and E10 embryos were permeabilized
with RIPA buffer and E12 embryos were treated with
10 ␮g/ml proteinase K for 15 min at room temperature
to enhance permeabilization. Following post-fixation
and prehybridization, the specimens were hybridized
overnight with 1 mg/ml DIG-labeled riboprobes at
70°C. The specimens were washed, blocked, and incubated with anti-DIG alkaline phosphatase conjugated
antibody (Boehringer Mannheim Corp., Indianapolis,
IN) at a dilution of 1:2000 for 90 min at 4°C. The
specimens were washed extensively after which endogenous alkaline phosphatase activities were blocked by
overnight incubation in 0.48 mg/ml levamisol (Sigma,
St. Louis, MO). The color reaction was developed using
nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl phosphate (BCIP) as substrates (Sigma, St.
Louis, MO). The area of gene expression induced by
BMP4-soaked beads and visualized by whole-mount in
situ hybridization was determined by first photographing the processed specimens and then subjecting the
images to morphometric analyses using NIH Image
Version 1.6.1 (NIH, Bethesda, MA). Signal to background was determined by density slice method within
412
SEMBA & NONAKA ET AL.
the software. Student’s t-test was performed on numerical data.
Semi-Quantitative RT-PCR
In order to maximize the sensitivity of detection,
multiple PBS- or 100 ng/␮l BMP4-soaked beads were
implanted at either the rostral or proximal positions on
one side of the E10 mandibular process and allowed to
grow in explant culture system. Twenty-four hr later,
each mandible was bisected along the midline in order
to separate the implanted half from the non-implanted
half. Four halves of the same treatment were pooled as
one sample and subjected to RT-PCR for Sox9, Msx2,
and beta-actin (Stratagene, La Jolla, CA). Amplimers
for Sox9 were 5⬘-AAGATAAGTTCCCCGTGTGC-3⬘ and
5⬘-GTAGTGAGGAAGGTTGAAGG-3⬘, and those for
Msx2 were 5⬘-TGTTTTCGTCGGATGAGGAG-3⬘ and
5⬘-GTCGCTTAGGGTGACAATGC-3⬘. Thirty, 28, and
18 cycles were empirically determined for Sox9, Msx2,
and beta-actin PCR, respectively to optimize for amplification linearity and signal. The expression levels of
Sox9 and Msx2 were normalized against beta-actin.
The results were subjected to Student’s t-test.
Adenovirus and Microinjection
Recombinant adenovirus carrying haemagglutin
(HA) epitope-tagged mouse Msx2 gene (AdV-Msx2)
(Takahashi et al., 1998b) or LacZ gene (AdV-LacZ) as
control were prepared. Ten nl of the recombinant adenovirus solution (titer of 1 ⫻ 1010 pfu/ml) accompanied
by 1% tetramethylrhodamine dextran (Molecular
Probes Inc., Eugene, OR), which was used as a visible
marker, were microinjected into the mandibular explants using a glass micropipette and a Transjector
5246 (Eppendorf, Hamburg, Germany) under a stereomicroscope. After 36 hr of culture, explants were
processed for HA immunostaining and ␤-galactosidase
staining as previously described (Takahashi et al.,
1998b) to confirm the site and spread of infection.
Quantitative RT-PCR for Type II Collagen
mRNA
Type II collagen mRNA expression levels were analyzed by quantitative RT-PCR method as previously
described (Takahashi et al., 1998a). Six explants were
pooled for each analysis. The amount of type II collagen
mRNA in the adenovirus microinjected half of the mandibular explants was compared with the control uninjected half for each AdV-LacZ and AdV-Msx2 infected
specimens after 6 days in culture and reported as their
relative ratio. The data were subjected to statistical
analyses using Student’s t-test.
ACKNOWLEDGMENTS
We are grateful to Dr. Yi-Hsin Liu (University of
Southern California) for mouse Msx2 cDNA, Dr. Silvio
Gutkind (National Institute of Dental and Craniofacial
Research) for AdV-LacZ and Ms. Audra Wright for
assistance with Figure 3. We also thank Dr. Hari Reddi
and Dr. Lee Niswander for valuable suggestions to this
project. Human recombinant BMP4 is a gift from Genetics Institute.
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