High frequency of cephalic neural crest cells shows
coexistence of neurogenic, melanogenic, and
osteogenic differentiation capacities
Giordano W. Calloni, Nicole M. Le Douarin1, and Elisabeth Dupin1
Centre National de la Recherche Scientifique Unite ´ Propre de Recherche 2197 Laboratoire De ´veloppement, Evolution et Plasticite ´ du Syste `me Nerveux,
Institut de Neurobiologie Alfred Fessard, 91198 Gif-sur-Yvette, France
Contributed by Nicole M. Le Douarin, April 7, 2009 (sent for review December 1, 2008)
The neural crest (NC) is a vertebrate innovation that distinguishes
vertebrates from other chordates and was critical for the devel-
opment and evolution of a ‘‘New Head and Brain.’’ In early
vertebrates, the NC was the source of dermal armor of fossil
jawless fish. In extant vertebrates, including mammals, the NC
forms the peripheral nervous system, melanocytes, and the carti-
lage and bone of the face. Here, we show that in avian embryos,
a large majority of cephalic NC cells (CNCCs) have the ability to
differentiate into cell types as diverse as neurons, melanocytes,
osteocytes, and chondrocytes. Moreover, we find that the mor-
phogen Sonic hedgehog (Shh) acts on CNCCs to increase endo-
chondral osteogenesis while having no effect on osteoblasts prone
to membranous ossification. We have developed culture condi-
tions that demonstrate that ‘‘neural–mesenchymal’’ differentia-
tion abilities are present in more than 90% of CNCCs. A highly
multipotent progenitor (able to yield neurons, glia, melanocytes,
myofibroblasts, chondrocytes, and osteocytes) comprises 7–13% of
the clonogenic cells in the absence and presence of Shh, respectively.
This progenitor is a good candidate for a cephalic NC stem cell.
clonal culture ? dermal bone ? endochondral bone ? multipotency ?
cells that appears at the border of the neural plate. CNCCs
cell types that include, in addition to chondrocytes and osteo-
cytes, diverse nonskeletal mesenchymal cells as well as neurons
and glial cells of the peripheral nervous system and pigment cells
(1). CNCC-derived osteoblasts yield either endochondral bones
(e.g., hyoid bone), which replace cartilaginous templates, or
membranous (dermal) bones (e.g., in skull vault), which differ-
entiate directly from mesenchymal condensations (2). How
skeletogenic cells become specified in the CNCC population and
whether they arise from multipotent progenitors need to be
further documented. Through previous in vitro clonal analysis of
avian CNCCs, a rare subset of common progenitors for chon-
drocytes and pigment, glial, and/or neuronal cells was identified
(3–6). Recently, we found that chondrocytes differentiate in
vitro from highly multipotent progenitors of NC origin (7), able
to yield both ‘‘neural’’ (i.e., neurons, glia, pigment cells) and
‘‘mesenchymal’’ (i.e., smooth muscle and connective cells, chon-
drocytes) cell types. The morphogen Sonic hedgehog (Shh), a
crucial factor for brain and face development (8–13), increased
the number of such progenitors in vitro (7). It has not yet been
established whether multipotent ‘‘neural–mesenchymal’’ ce-
phalic NC (CNC) progenitors can also give rise to osteocytes, the
alternative being that osteogenic cells constitute a separate
lineage within the CNCC population. It is also unknown whether
the skeletogenic cells that form endochondral and dermal bones
in the head derive from common CNC progenitors.
Based on the expression of Runx2, a master transcription
ost of the skull of vertebrates is derived from the cephalic
neural crest cells (CNCCs), a population of embryonic
we have analyzed the osteogenic properties of quail CNCCs in
mass and clonal cultures and investigated the effect of Shh on the
development of skeletal progenitors. We describe that osteo-
blasts differentiate in CNCC cultures either at a distance from,
or closely associated with, the perichondrium surrounding car-
tilage nodules. Only the perichondrial endochondral-like osteo-
blasts, but not those unconnected to chondrogenic islets, showed
enhanced differentiation and proliferation in presence of Shh. In
single-cell culture, the great majority of clone-forming CNCCs
were capable of generating osteoblasts together with cells of the
neural and melanocytic lineages, and all of the osteogenic
progenitors were multipotent (or at least bipotent). Moreover,
we identify an NC progenitor yielding neurons, glia, pigment
cells, myofibroblasts, chondrocytes, and osteoblasts, which has
never been described so far and could be assimilated to a highly
multipotent stem cell in the early CNC.
Differentiation of Runx2?Osteoblastic Cells in CNC Cultures and
Influence of Shh on Perichondrial Osteoblasts. With the aim of
analyzing the osteogenic capacity of CNCCs in vitro, we used
culture conditions that had been proven in our previous inves-
tigations to be suitable for differentiation of CNCCs into various
mesenchymal phenotypes (7). Briefly, mes-rhombencephalic
15 h of primary culture of the neural primordium, and after 6
days of subculture they generated mesenchymal (i.e., myofibro-
blasts/smooth muscle cells and chondrocytes) and nonmesen-
chymal, neural cell types (i.e., neurons, glia, and melanocytes).
Because in these experiments addition of Shh during the first
48 h of culture vigorously promoted chondrogenesis, we have
investigated the osteogenic properties of CNCCs grown in the
absence and presence of 100 ng/mL recombinant Shh.
To recognize the cells engaged in the osteogenic differentia-
tion pathway, we examined the expression of Runx2, a transcrip-
tion factor required for endochondral and membrane bone
formation in vivo (14–16) that is expressed in avian craniofacial
skeleton from embryonic day 6.5 (E6.5) onward (17, 18). In day
10 (d10) cultures, we identified 3 types of osteoblastic CNCCs:
nodules, designated as perichondrial cells (Fig. 1C), (ii) Runx2?
cell islands not associated with chondrocytic aggregates (Fig. 1I),
and (iii) isolated Runx2?cells located randomly (Fig. 1G).
Continuous treatment with Shh from d0 to d10 increased by 50%
the percentage of perichondrial areas that expressed Runx2
compared with control cultures (Fig. 1 A–C). To investigate
whether Shh acted during a particular window of time, we
Author contributions: G.W.C., N.M.L.D., and E.D. designed research; G.W.C. and E.D.
performed research; G.W.C., N.M.L.D., and E.D. analyzed data; and G.W.C., N.M.L.D., and
E.D. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: email@example.com
June 2, 2009 ?
vol. 106 ?
no. 22 ?
submitted CNCC cultures to Shh treatment from d0 to d7, from
d7 to d10, or from d0 to d10 as described above (Fig. 1A). Similar
to continuous exposure to Shh, treatment with Shh between d7
and d10 significantly increased the number of perichondrial
areas that expressed Runx2 at d10 compared with controls (Fig.
1 A–C). The positive effect of Shh on perichondrial osteoblast
differentiation took place between d7 and d10. Accordingly,
addition of both Shh and the alkaloid cyclopamine, an inhibitor
of Shh signaling (19), from d7 to d10 blocked Shh-mediated
increase of Runx2 perichondrial expression (Fig. 1D).
In addition to its requirement for osteogenesis, Runx2 plays a
role in chondrocyte maturation and hypertrophy (20). There-
fore, we looked for expression of both Runx2 and Sox9 tran-
scription factors, because the latter is activated in vivo specifi-
cally in chondrocytes (21, 22). The chondrocyte nodules only
expressed Sox9, whereas Runx2 expression was detected mainly
in the perichondrium in d10 cultures (Fig. 1E). Runx2?osteo-
blastic cells and Sox9?chondrocytic cells thus formed nonover-
lapping, adjacent cell populations.
We also found that Runx2 was expressed in d10 cultures
independently of the presence of cartilage nodules, in either
isolated cells (Fig. 1G) or cell islands (Fig. 1I). Quantification of
Runx2?isolated cells and of islands expressing Runx2 showed no
difference between Shh-treated and untreated cultures (Fig. 1 F
and H). Moreover, addition of both cyclopamine and Shh did not
change the amount of Runx2?isolated cells and cell islands
recorded in the cultures (Fig. 1 F and H).
Effect of Shh Treatment on Gli Expression and Cell Proliferation in
Runx2-Expressing CNCCs. To gain further insight about the role of
Shh in osteogenesis by cultured CNCCs, we first examined the
expression of the Gli genes encoding Shh signaling effector
proteins Gli1, Gli2, and Gli3 (23). In d10 cultures exposed to Shh
from d7 to d10, we observed an increase in Gli2 expression in
perichondrial regions compared with control cultures and with
those treated from d0 to d7 (Fig. 2 A and B). We did not detect
Gli1 and Gli3 transcripts in either culture condition. Therefore,
in the perichondrium, enhancement of Gli2 by Shh coincides
with the increase in Runx2, suggesting that Gli2 mediates the
effect of Shh on the development of perichondrial osteoblasts.
To examine osteoblast proliferation, we performed a pulse
labeling with BrdU at d10. CNCC cultures exposed to Shh
exhibited a higher proportion of Runx2?perichondrial cells
incorporating BrdU, whereas the proportion of Runx2?isolated
influences perichondrial osteoblasts. Runx2 expression was studied by in situ
hybridization in d10 cultures of CNCCs grown in the absence and presence of
cyclopamine during 3 different time periods: d0–d10, d0–d7, and d7–d10.
The total number of perichondrial areas and the number of those expressing
d7–d10 treatment increase the percentage of perichondrium containing
Runx2?cells; this effect of Shh is blocked by cyclopamine (n indicates the
of Shh (C), or in the presence of both Shh and cyclopamine (D). (E) Double in
situ hybridization showing Sox9 (red) and Runx2 (green) expression in chon-
drocytes and osteoblasts, respectively (blue shows Hoechst nuclear staining).
(F–I) Nonperichondrial Runx2?cells are isolated (G) or grouped in cell islands
(I); the number of Runx2?isolated cells (F) and the number of cell islands
expressing Runx2 (H) do not change in the different media (F and H; n ? 12).
Data are shown as mean (?SEM) of 3 independent experiments. (Magnifica-
tions: B–E and I, 55?; G, 220?.)
Runx2-expressing osteoblasts differentiate in CNCC cultures; Shh
expression increased in CNCC cultures treated with Shh from d7 to d10 (B)
compared with d0–d7 treatment (A), similarly to Runx2 (Fig. 1A). (Magnifi-
cation: 80?.) (C) BrdU incorporation and Runx2 expression in Shh-treated
(d7–d10) and untreated d10 cultures; the proportion of BrdU-incorporating
Runx2?cells is indicated for Runx2?perichondrial cells, Runx2?isolated cells,
and Runx2?cell islands. Data are given as mean (?SEM) of 3 independent
experiments (n ? 12 cultures).*, P ? 0.05.
Shh promotes Gli2 expression and proliferation of Runx2?cells. Gli2
www.pnas.org?cgi?doi?10.1073?pnas.0903780106Calloni et al.
cells and Runx2?cells in islands, which were labeled with BrdU,
was unchanged compared with control cultures (Fig. 2C).
Expression of Bone Matrix Proteins by Differentiated Osteogenic
CNCCs. To assess whether the osteogenic CNCCs that develop in
the perichondrium in the presence of Shh are able to acquire
mature ossification markers, Shh-treated cultures were further
grown from d7 to d15 in the presence of an osteogenic medium
(24). These conditions triggered synthesis of the bone matrix
proteins collagen-1?1, bone sialoprotein, and osteonectin in the
perichondrial areas (Fig. 3 A–C) and promoted mineral depo-
sition, as detected by Alizarin red staining (Fig. 3D).
Characterization of Osteoblastic Progenitors in CNCC Clonal Cultures.
To identify osteogenic progenitors, we performed clonal cul-
tures of CNCCs in the absence and presence of Shh from d7 to
d10 (as described above as promoting perichondrial osteogen-
esis). Quantification of d10 colonies showed that the clonal
efficiency of CNCCs slightly increased in Shh-treated cultures
compared with untreated cultures [65% (n ? 146) and 52% (n ?
109), respectively; P ? 0.04]. We analyzed the CNCC clonal
progeny from the main NC-derived cell types by using pheno-
typic markers (Fig. 4A). Runx2?osteoblasts differentiated in
nearly all colonies in both control (94%) and Shh-supplemented
(96%) medium. Addition of Shh increased the frequency of the
clones containing chondrocytes (from 20% to 50%) and myo-
fibroblasts (from 54% to 78%) compared with control medium
(Fig. 4A). According to the combinations of cell types they
contained, 30 distinct clone types were recorded (Fig. 5). No-
ticeably, we identified a highly multipotent GNMFCO progen-
itor able to give rise to all of the expected phenotypes (glial,
neuronal, melanoblastic, myofibroblastic, chondrocytic, and os-
teoblastic; Fig. 4 B–E), which exhibited a higher frequency in the
presence of Shh (13%) than in controls (7%; P ? 0.009; Fig. 5).
The majority of the other progenitors in both media were
pentapotent, quadripotent, and tripotent CNCCs endowed with
osteoblastic potential. The most frequent in Shh-treated cultures
corresponded to a GMFCO pentapotent progenitor (23%), a
progenitor very rare in control conditions (0.5%). In contrast,
Shh decreased the frequency of GNMO and GMO progenitors
2.5-fold and 4-fold, respectively (Fig. 5).
Three kinds of skeletal progenitors were identified in these
experiments: (i) O?C?osteochondrogenic progenitors, (ii)
O?C?progenitors yielding osteoblasts but no chondrocytes, and
(iii) O?C?progenitors generating chondrocytes but no osteo-
blasts. The last group of chondrogenic CNCCs was recorded at
a low frequency in both media. By contrast, the osteochondro-
genic progenitors increased and the osteogenic progenitors
decreased significantly in the presence of Shh (Fig. 5). There-
fore, although it did not modify the overall frequency of osteo-
genic CNCCs (Fig. 4A), Shh favored the development of osteo-
chondrogenic progenitors at the expense of those able to yield
osteoblasts but no chondrocytes. Taken together, because of the
progenitors accounted for more than 92% of clonogenic cells in
both control and Shh-supplemented media (Fig. 5, gray progen-
itors). Hence, the proportion of CNCCs with exclusively neural
or mesenchymal potentials (Fig. 5, yellow and blue, respectively)
was very low in both conditions.
In contrast to the trunk skeleton, which derives from mesoder-
mal cells, most of the head skeleton arises from the CNC in
higher vertebrates. CNCCs give rise to intramembranous bones
in the skull vault, the otic capsule and jaw, and to endochondral
cultures maintained from d7 to d15 in osteogenic medium (see Materials and
Methods) comprise perichondrial cells immunoreactive to collagen-1?1
(COL1A1) (A), bone sialoprotein 1 (BSP) (B), and osteonectin (OSN) (C) and
which stain with Alizarin red (D). (Magnification: 50?.)
Cultured CNCCs express bone matrix proteins. Shh-treated CNCC
analyzed by in situ hybridization and immunocytochemistry to detect osteo-
blasts, chondrocytes, myofibroblasts, melanocytes, neurons, and glial cells
phenotype. Nearly all colonies include osteoblasts; Shh increases the total
expressed as mean percent ? SEM from 4 independent experiments (n ? 109
P ? 0.01;*, P ? 0.05). (B–E) GNMFCO multiphenotypic colony including: (B)
Runx2?osteoblasts, (C) ?-SMA?myofibroblasts (red) and HNK1?glial cells
arrow next to the glial cells), and (E) melanocytic cells (MelEM?in green, next
to the neurons). Asterisks in B–D indicate cartilage nodules. (Magnification:
Calloni et al.PNAS ?
June 2, 2009 ?
vol. 106 ?
no. 22 ?
bones, such as the nasal capsule and the quadrate and hyoid
bones, which originate from a cartilage rudiment (1, 25, 26).
Despite the importance of the CNC in building craniofacial
structures, little is known about the emergence and specification
of skeletogenic NC progenitors. Here, we have characterized the
developmental potentials and response to Shh of osteogenic
CNCCs in vitro.
CNCCs Differentiate in Vitro into Perichondrial and Dermal-like Os-
teoblasts. By exploiting a culture system that previously allowed
quail CNCCs to develop along the main NC-derived lineages,
including the chondrocytic lineage (7), we have characterized in
vitro osteoblastic differentiation of CNCCs isolated at early
migratory stages from the mes-rhombencephalon of 6–7 somite-
stage quail embryos. Osteoblastic cells, as defined by expression
of the early marker gene of osteogenesis Runx2 (14–16), were
detected from d7, increased in number at d10, and were still
present in d15 cultures. This is consistent with the temporal
pattern of Runx2 described in vivo (17, 18).
Runx2?cells were found in association with chondrocyte
nodules or in chondrocyte-free areas of the cultures as isolated
cells or grouped in cell islands. The first type of osteoblasts
differentiated in the perichondrium of cartilage nodules that had
formed at earlier stages of CNCC culture (7). When CNCCs
were further maintained in medium conditions promoting os-
teogenesis (24), the perichondrial osteoblasts underwent syn-
thesis of bone matrix proteins and showed mineral deposits. In
contrast, the Runx2?isolated cells and cell islands in these
cultures did not reach the matrix-secreting mature state. Based
on their differential bone marker expression and distinct loca-
tions relative to chondrocyte nodules, the CNCC-derived peri-
chondrial osteoblasts can be considered as undergoing endo-
chondral-like ossification (27, 28), whereas the Runx2?cell
islands can be assigned to a dermal-like type of osteoblast, which
does not require a cartilage template to develop (17, 18, 29).
Shh Regulates in Vitro Proliferation and Differentiation of the CNC-
Derived Perichondrial Osteoblasts. Differences between the types
of Runx2?osteoblasts recorded in CNCC cultures are further
exemplified by their differential response to Shh. When added to
CNCC cultures from d7 to d10 (once cartilage nodules have been
formed), Shh has a critical role in promoting differentiation and
proliferation of Runx2?perichondrial cells. This result is con-
sistent with the ability of Shh to trigger osteogenic differentia-
tion in mesenchymal and preosteoblastic cell lines (30–32). The
Shh-induced increase in perichondrial osteoblasts was blocked
by cyclopamine and correlated with up-regulation of perichon-
drial Gli2 expression, suggesting that Shh acts on perichondrial
osteoblasts in CNCC cultures through Smoothened and Gli2
effector proteins. This possibility is supported by the osteopenic
phenotype of Gli2 knockout mice (33) and by in vitro studies
showing that Gli2 mediates Shh action on Runx2 expression and
osteoblast differentiation by mesenchymal cells (34, 35). In
contrast to perichondrial osteoblasts, we found no detectable
effect of Shh on dermal-like osteoblasts, which differentiated
added continuously or from d7 to d10). Shh treatment did not
up-regulate Gli2 expression in Runx2?cell islands.
Taken together, these findings are consistent with in vivo data
showing that Hedgehog signaling mediated by Indian hedgehog
(Ihh) is indispensable for endochondral skeleton development
(36–39). Although reduced, membranous ossification occurs in
the skull of mice deleted for Ihh (39). Therefore, compared with
endochondral ossification, a distinct, hedgehog-independent
mechanism likely regulates osteoblast commitment in membra-
nous bones in vivo and in CNCC cultures.
Differential Responsiveness to Shh Signaling. In single-cell cultures,
more than 90% of clonogenic CNCCs turned out to be able to
yield osteoblasts. Nearly all of these osteogenic CNCCs were
multipotent progenitors with diverse combinations of neural and
mesenchymal differentiation potentials. According to their abil-
ity to generate chondrocytes and/or osteoblasts, skeletogenic
multipotent CNCCs were identified as osteochondrogenic, os-
teogenic only and, less frequently, chondrogenic only. These
in a schematic lineage tree in which progenitor types are classified according to the number of cell types in their progeny (G, glial cells; N, neurons; M,
melanocytes; F, myofibroblasts; C, chondrocytes; and O in red, osteoblasts). The frequency of each progenitor type (percent of clones) is shown in both medium
(i.e., F, C, O) and neural (i.e., G, N, M) potentials (in gray) are widespread compared with those yielding only neural (in yellow) or only mesenchymal (in blue)
Osteogenic CNCCs are multipotent and the targets of Shh. The analysis of control (n ? 109) and Shh-treated (n ? 146) colonies (Fig. 4) is summarized
www.pnas.org?cgi?doi?10.1073?pnas.0903780106 Calloni et al.
types of skeletogenic progenitors strikingly recapitulate the 3
distinct skeletal structures arising from the CNC—i.e., endo-
chondral bones, dermal bones, and persistent cartilages (18).
These results have several implications for skeletogenic cell
lineage segregation in the CNC. First, the CNCCs do comprise
common progenitors for bone and cartilage, designated as
osteochondrogenic progenitors, the existence of which was
inferred by genetic fate mapping (40). Second, our finding that
most CNCCs can generate osteoblasts but no chondrocytes
strongly argues that dermal bone cells can arise from specific
progenitors distinct from the osteochondrogenic, endochondral-
like ones. Hence, the endochondral and dermal bone cell
CNCCs that can be distinguished by their response to Shh; this
factor specifically favors osteochondrogenic CNCCs (O?C?) at the
(Fig. 5). This action of Shh on clonogenic CNCCs is consistent with
the effects of Shh observed in mass cultures, in which Shh stimu-
lated the development of perichondrial, endochondral-like osteo-
located in cell islands. The increase in osteochondrogenic progen-
during the first 48 h of culture (7, 41).
An Shh-Responsive, Multipotent Osteochondrogenic Progenitor Lies
Upstream of CNCC Hierarchy. We identified a highly multipotent
CNCC that generated a progeny containing glial cells, neurons,
melanocytes, myofibroblasts, chondrocytes, and osteoblasts
(GNMFCO). This hexapotent progenitor lies upstream of all of
the other NC progenitors described thus far (42). It is a good
candidate for being the NC stem cell comparable to the hema-
topoietic stem cell able to yield all of the blood cell types. For
deserving this status, however, it lacks the proven self-renewal
capacity fully demonstrated for the hematopoietic stem cell (43).
The GNMFCO progenitor responds to Shh treatment by a 2-fold
increase in frequency, arguing that Shh regulates the survival of
multipotent CNCCs, as shown in our previous work for the
GNMFC progenitor (7). Taken together, the pentapotent and
hexapotent progenitors identified in the present experiments
accounted for 51% of total clones recorded in the presence of
Shh, compared with 23% in the absence of Shh (Fig. 5). These
results suggest that Shh regulates the survival and proliferation
of multipotent stem cells in the CNC, as observed in the enteric
nervous system (44, 45) and CNS (46–48). Finally, in both
control and Shh-treated cultures, more than 90% of CNCC
progenitors were endowed with neural–mesenchymal potential-
ities, whereas progenitors of exclusively neural or mesenchymal
phenotypes were very rare. This reflects the high proportion of
most frequent representatives of the neural and mesenchymal
NC lineages, respectively.
The present results thus uncover a previously unsuspected prev-
alence of the osteogenic potential in the early CNC, which may be
related to the notion drawn from fossil data that, in all likelihood,
such as ostracoderms (49). Subsequently, an internal skeleton of
mesodermal origin replaced the exoskeleton, permitting a signifi-
cant increase in body mobility. The skeletogenic capacities of the
NCCs were retained in the dorsal fin of teleosts and in the cranial
and facial skeleton, even in the most evolved vertebrates. The
capacity to yield osteocytes and chondrocytes together with glia,
neurons, and melanocytes, which is present in single CNCCs, thus
might be a striking remnant of the initial role played by the NC
during vertebrate evolution.
Materials and Methods
Cell Cultures. CNCCs were isolated from the neural primordium (mes-
rhombencephalon) of 6–7 somite-stage quail embryos. After 15 h of primary
culture, CNCCs that had migrated from explanted neural tubes were har-
vested for secondary plating on a feeder layer of growth-inhibited 3T3 fibro-
plates (TPP). Control culture medium was DMEM containing 10% FCS. In
Shh-treated cultures, the medium was supplemented with 100 ng/mL mouse
N-Shh (R & D Systems) during the whole culture period (d0–d10), during only
the first 7 days (d0–d7), or from d7 to d10 (d7–d10). In the last of these
conditions, an initial treatment during the first 48 h of culture (d0–d2) was
also performed, which enhanced differentiation into cartilage (7, 41) but did
not affect osteogenesis by CNCCs (Fig. 1A). In clonal cultures, 2% chicken
embryo extract was added to the medium. Inhibition of Shh signaling was
performed by treatment with 5 ?M cyclopamine (19) (Toronto Research
d15 in osteogenic medium (24)—i.e., control medium supplemented with 10
nM dexamethasone, 1 mM ?-glycerol phosphate, and 50 ?g/mL ascorbic acid
(all from Sigma). Cultures were maintained at 37 °C in a humidified 5%
CO2/95% air atmosphere.
Phenotype Analysis. Cultures were fixed with 4% paraformaldehyde at d10 or
d15. Quail CNCCs were distinguished from mouse 3T3 fibroblasts by Hoechst
hybridization with a digoxygenin-labeled probe for chicken Runx2 [gift from T.
Jaffredo,CentreNationaldelaRechercheScientifiqueUnite ´ MixtedeRecherche
7622, Paris, France] according to an already described procedure (7). Differenti-
ation of chondrocytes in 3-dimensional nodules was assessed by phase-contrast
microscopy and after in situ hybridization for chick Sox9 (50) as described previ-
ously (7), except that a fluorescein-labeled RNA probe was revealed by using a
Tyramide System Amplification Kit (Invitrogen). Bone matrix proteins were de-
tected by using antisera against bone sialoprotein-1 (BSP; LF-84, LF-119; 1:100),
osteonectin (LF-8, LF-45; 1:200), and collagen-1?1 (LF-67; 1:100), a generous gift
from L. W. Fisher (51) (National Institute of Dental and Craniofacial Research,
skeletal phenotypes was performed essentially as described previously (7) by
using the following antibodies: Melanoblast/melanocyte Early Marker (MelEM)
Sigma) for myofibroblasts/smooth muscle cells, and Tubulin ?III (5G8; Promega)
and tyrosine hydroxylase for neurons and adrenergic cells, respectively. Second-
ary antibodies were purchased from Southern Biotechnology Associates. Cell
proliferation activity was analyzed by BrdU incorporation (1-h pulse before
fixation) detected by immunofluorescence using a Cell Proliferation Kit (Roche).
Fluorescence was observed with an X70 Olympus microscope. Statistical analysis
(for clone frequency). Differences between control and Shh-treated cultures
were considered significant when P ? 0.05.
ACKNOWLEDGMENTS. We thank L. W. Fisher for the gift of the antisera to
acknowledge A. Gonc ¸alves-Trentin (Federal University of Santa Catarina,
Santa Catarina, Brazil) and J. S. Joly [Centre National de la Recherche Scien-
tifique (CNRS) Unite ´ Propre de Recherche 2197] for helpful support to G.W.C.
This study was supported by CNRS and Association pour la Recherche sur le
Cancer (ARC; Grant N3929). G.W.C. was supported by an ARC postdoctoral
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