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First published online June 6, 2008
doi: 10.1242/10.1242/dev.019125
1 CNRS, UPR 2197, Laboratoire de Développement, Evolution et
Plasticité du Système nerveux, Institut de Neurobiologie Alfred
Fessard, F-91198 Gif-sur-Yvette, France.
2 CNRS, UMR 7622, Laboratoire de Biologie du Développement, Paris,
France.
3 UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement,
Paris, France.
* Author for correspondence (e-mail: nicole.ledouarin{at}academie-sciences.fr)
Accepted 6 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Neural crest, Chick embryo, BA1, Meckel's cartilage, Shh, Bmp4, Fgf8, Lower jaw
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). By constructing quail-chick chimeras in ovo, it was shown
that NCCs, which exit from the diencephalon posterior half down to rhombomere
2, colonize the facial buds that are at the origin of the upper beak [derived
from the nasofrontal and nasolateral together with the maxillary buds (Mx)],
and the lower jaw [derived from the mandibular buds (Md)]
(Couly et al., 1996;
Köntges and Lumsden,
1996; Lee et al.,
2004; Cerny et al.,
2004).
It has been shown in our laboratory that duplication of the lower-jaw
skeleton (composed of Meckel's cartilage and associated membrane bones) can be
induced by grafting a fragment of ventral foregut endoderm removed from a
definite area of 6-somite stage (6 ss) quail embryos, at the presumptive level
of the first branchial arch (BA1) in stage-matched chicken
(Couly et al., 2002). Similar
Meckel's cartilage duplication was obtained before by grafting Bmp4-soaked
beads into Md at embryonic day 3.5 (E3.5)
(Barlow and Francis-West, 1997;
Mina et al., 2002) and more
recently by replacing Md ectoderm by the so-called `frontal ectodermal zone'
(FEZ) (Hu et al., 2003).
In a precedent work (Brito et al.,
2006), we demonstrated that Sonic Hedgehog (Shh)
expression in the ventral foregut endoderm, from the early somitic stages
onwards, is crucial for the survival and further development of the cephalic
NCCs that colonize BA1. Excision of the presumptive forehead area, including
the precordal plate (PcP) and the anterior-most Shh-expressing
foregut endoderm, definitively impairs Shh expression in BA1 foregut
endoderm. Although mesencephalic NCCs migrate normally to BA1 under these
conditions, their survival does not ensue and lower jaw does not develop. The
forehead, which contains Shh-producing structures at that stage (PcP and
anterior ventral endoderm), can be substituted for by exogenous Shh provided
through a heparin bead applied to the section surface, hence in contact with
the ventral foregut endoderm. In such a case, BA1 ventral endoderm starts to
express Shh and a lower jaw develops. The crucial role of Shh on
facial skeleton development is in line with the effect of Shh gene
inactivation in the mouse, where holoprosencephaly, cyclopia and complete
absence of facial skeleton (including lower jaw) were observed, in spite of
the presence of BA1 at E9.5 (Chiang et al.,
1996). Part of those phenotypes were also obtained by grafting
cells producing antibodies against Shh in the presumptive facial area of 7-14
ss chick embryos (Ahlgren and
Bronner-Fraser, 1999), and also by conditional knockout of the
Smoothened (Smo) gene in the cephalic NCCs
(Jeong et al., 2004). By
contrast, overexpression of Shh in Md ectoderm of E2 chick embryos
resulted in the formation of ectopic cartilage that branched off from the
endogen Meckel's cartilage (Haworth et
al., 2007).
In order to document further the role of Shh in BA1 patterning and
lower-jaw development, we have tested the effect of this morphogen
administered to the presumptive territory of BA1 in 5-8 ss chick embryos,
through grafting quail fibroblasts of the QT6-line engineered to secrete Shh
[QT6-Shh cells (Duprez et al.,
1998)]. Surprisingly, the presence of an exogenous source of Shh
in BA1 mesenchyme, resulted in the induction of supernumerary Meckel's
cartilages, which develop in mirror-image orientations, caudolateral to the
normal jaw. Development of these structures is preceded by specific
heterotopic gene activities in BA1 caudal region. This process is strikingly
similar to the molecular events associated with digit duplication resulting
from the transfer of posterior limb bud mesenchyme (zone of polarizing
activity or ZPA), or of Shh-producing cells, into the anterior region of the
developing limb bud (Saunders and
Gasseling, 1968; Riddle et
al., 1993).
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
maintained in the standard culture medium (DMEM supplemented with 10% FBS and
selecting antibiotic G418). Cells to be grafted were prepared as described
before (Teillet et al., 1998).
Twenty-four hours before the graft, they were seeded (1x106
cells/ml standard medium without G418) into (60 mm) bacteria plates not
treated for cell adhesion. Cell aggregates were transferred and grafted into
chick embryos in ovo (Gallus gallus, JA57, ISA-France). The fate maps
of the head region constructed by Couly served as landmarks
(Couly and Le Douarin, 1985;
Couly and Le Douarin, 1990).
Operated embryos were reincubated at 38°C under humidified and ventilated
atmosphere until they reach E3-4 for whole-mount, E4-6 for immunochemistry or
in situ hybridization on sections, or E10-12 for morphology and skeleton
studies.
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In situ hybridization of whole mounts and sections
Embryos at E3-6 were fixed in 4% formaldehyde in PBS. We used the
whole-mount in situ hybridization procedure of Henrique et al.
(Henrique et al., 1995).
Antisense RNA probes were synthesized as described previously for
Fgf8 (Crossley et al.,
1996), Shh (Riddle et
al., 1993), Bmp4
(Francis et al., 1994),
Pitx1 (Henrique et al.,
1995), Dlx5 (Pera et
al., 1999), MyoD
(Pourquié et al.,
1996), dHand (Howard
et al., 1999) and Sox9
(Kordes et al., 2005).
Skeletal staining
Embryos at E10-11 were fixed overnight in acetic alcohol containing 15%
Alcian Blue. Bones were stained using Alizarin Red in alcohol and soft tissues
were cleared with 1% KOH in 20% glycerol
(Ojeda et al., 1970).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
grafted in the right BA1 presumptive territory of 5-8 ss chick embryos in ovo
(Fig. 1A, circle). Seven hours
after the graft, at 10 ss, QT6-Shh cells, revealed by in situ hybridization
with a Shh probe, were found in close contact with the host BA1
ectoderm and pharyngeal endoderm (Fig.
1B). At E7-11, either one (n=2/12) or two
(n=7/12) hemi-lower-beaks had ectopically developed caudal to the
normal jaw on the operated side (Fig. 1C,
1-3). Other embryos (n=3/12) looked normal. Generally,
the supernumerary structures were similar in size and shape to the endogenous
lower beak. Interestingly, they looked like a series of mirror images
(Fig. 1C). Analysis of the
skeleton by Alcian Blue and Alizarin Red staining revealed the presence of
recognizable cartilage and bone elements derived from BA1. The supernumerary
Meckel's cartilages had their proximal part associated with the normal
Meckel's cartilage (Fig. 1D,
arrow), thus forming a large structure from which two or three separate
branches emerged (Fig. 1E,
1-3). The disposition of Md bones (dentary and splenial), present
in all ectopic beaks, confirmed the mirror-image orientation
(Fig. 1E,F, arrows). By
contrast, the proximal (dorsal) cartilages of the lower jaw (quadrate and
articular) were not duplicated and showed normal shape and size
(Fig. 1D, arrowhead). In the
non-grafted side, no supernumerary element developed (not shown). Bilateral grafts of QT6-Shh cells were also performed (see Fig. S1 in the supplementary material). Out of 25 operated embryos, only one survived up to E10. In this only case, the supernumerary beaks on the right side were similar in size and shape to the normal one and presented mirror images as described for right unilateral grafts (see Fig. S1B-D in the supplementary material). By contrast, on the left side, a small bifurcated ectopic beak developed, showing some cartilage and bone elements (see Fig. S1D-F in the supplementary material).
Grafts of control QT6 cells not carrying the Shh construct, were not capable of inducing supernumerary skeletal structures (n=4) and did not affect the jaw morphology (see Fig. S2A-C in the supplementary material).
These experiments show that an extra source of Shh applied in the BA1 presumptive territory before NCC migration can trigger supernumerary lower-jaw development without interfering with the growth and orientation of the normal jaw.
Grafted QT6-Shh cells remain in the proximal BA1
In order to obtain further information on the mechanism of action of Shh in
lower-jaw development, we traced the grafted QT6-Shh cells in embryos operated
unilaterally on the right side, both through their content of Shh
transcripts and by using the quail-specific QCPN mAb. Three days after the
graft, at E4, BA1 was hypertrophied on the grafted side with an abnormal
posterior Md extension (Fig.
2A,B, arrow). Frontal serial adjacent sections were alternatively
immunostained with QCPN or hybridized with a Shh probe
(Fig. 2B,C-E'). In the
four cases analyzed, no QT6-Shh cells were found in the distal Md
(Fig. 2C,C'), only very
few of them, which were QCPN positive, remained in the medial part of Md
prominence (Fig. 2D,D',
arrowheads) and most of the QCPN-positive cells, which also contained
Shh transcripts, were localized within Md proximal half
(Fig. 2E,E',E'').
Thus, although devoid of QT6-Shh cells, the distal part of BA1 exhibited both
hyperplasia and the onset of duplication process
(Fig. 2C,C';
Fig. 5E), suggesting that Shh
might act indirectly on BA1 outgrowth and development. Next observations
showed that Shh action is primarily mediated through gene expression pattern
modifications in the developing BA1 ectoderm.
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; Wall and Hogan,
1995; Trumpp et al.,
1999; Creuzet et al.,
2004; Haworth et al.,
2004; Liu et al.,
2005). In E3 and E4 chick embryos grafted with QT6-Shh cells, both
Fgf8 and Bmp4 transcripts were also present in the caudal
region of BA1 ectoderm on the grafted side
(Fig. 3A,C, arrows) where none
of them is normally expressed, as seen on the contralateral non-grafted side.
Therefore, Shh-secreting cells induced an ectopic caudal expression of
Fgf8 and Bmp4, thus affecting BA1 gene expression pattern
along its rostrocaudal axis. Moreover, Shh itself was induced
ectopically in BA1 caudal ectoderm (Fig.
3B, arrows), whereas it is normally expressed only in BA1 endoderm
(Fig. 3B)
(Brito et al., 2006). These
extra sites of Fgf8 and Bmp4 expression led us to focus our
attention on Noggin, the expression of which can be induced by Bmp4
in Md mesenchyme (Stottmann et al.,
2001). We observed, in whole-mount E4 embryos, an abnormal site of
Noggin expression (n=4) located mediodistally in the caudal
part of the grafted BA1 (Fig.
4A, arrow), in a region where Bmp4 expression is also
abnormally present (Fig. 3C,
arrows).
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),
was not expressed in BA2 mesenchyme, but only in the ectoderm at the BA1/BA2
limit (see Fig. S3E4,F4, arrow in the supplementary material). By contrast,
distinct domains of Pitx1 expression could be distinguished in
experimental BA1 mesenchyme (Fig.
3F; see Fig. S3C4-F4 in the supplementary material).
Pitx1 whole-mount in situ hybridization at E4 confirmed this
observation showing its caudolateral extension in BA1
(Fig. 4B, arrow). This showed
that QT6-Shh grafts did not endow BA2 with BA1 properties. Notably, BA2 still
normally expressed Hoxa2 in these embryos (data not shown). This indicated
that the supernumerary structures were exclusively derived from BA1. Thus, the presence of an extra Shh signal in BA1 mesenchyme was responsible for the ectopic expression of Shh, Bmp4 and Fgf8 in caudal BA1 ectoderm, which exhibited a fold separating two Mds (Fig. 3H arrow). As schematized in Fig. 8C,C', the expression patterns of these genes, in relation with the ectodermal fold, probably determines the rostrocaudal orientation of the supernumerary lower jaws observed later and consequently their mirror-image polarity.
This prompted us to examine Gli3 expression, the loss of function
of which was implicated in polydactylism
(Hui and Joyner, 1993;
te Welscher et al., 2002a;
Littingtung et al., 2002). Gli3 can act as a transcriptional activator of
hedgehog target genes, or as a repressor in absence of Shh signaling (for a
review, see Ruiz i Altaba,
2006). Moreover, Gli3 expression in the limb bud, has
been shown to be controlled by dHand and reciprocally
(te Welscher et al., 2002b),
and dHand misexpression in the anterior limb bud resulted in ectopic
Shh expression and mirror-image digit duplication
(Charité et al., 2000;
Fernandez-Teran et al., 2000;
McFadden et al., 2002). In our
experiments, we observed that normal Gli3 expression in the medial
BA1 posterior half at E3 became considerably reduced after the graft of
QT6-Shh cells (Fig. 4C, arrow).
By contrast, dHand transcripts, which are normally present only in
the distal BA1 along its entire rostrocaudal axis, showed a lateral expansion
in its caudal part, which coincides with absence of Gli3 expression
(Fig. 4C,D).
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Thus, QT6-Shh grafts induce Shh, Fgf8 and Bmp4 expression in the caudal region of BA1 ectoderm leading to `rostralization' of the ectoderm and mesenchyme of the caudal-most part of BA1.
Shh triggers the program of lower-jaw development
We observed that ectopic expression of Bmp4 and Fgf8
induced by QT6-Shh cells in BA1 caudal ectoderm at E3-4 resulted, at E5, in
the growth of caudal expansions lined at their tip by a thickened ectodermal
layer still expressing Bmp4 (Fig.
5A,B, arrows). This thickening recalls the apical ectodermal ridge
of the limb bud as already described in other experiments
(Richman and Tickle, 1992).
Moreover, Sox9 (Fig.
5C) and collagen 2A1 (Col2a1)
(Fig. 5F), expressed in
precartilage nodules of NCC origin, were largely expanded caudally and
distally in the grafted BA1. At the same time, Dlx5 and
Pitx1 areas of expression were extended in BA1 overgrowth in contact
with the Bmp4-expressing ectoderm
(Fig. 5G-I, arrows). At E6, in
the normal mandible, Cbfa1, a master gene for ossification, was
expressed around Col2a1-expressing Meckel's cartilage and in contact
with the ectoderm (Fig. 6C,D).
Similarly, in the ectopic mandible, Cbfa1 mRNA was present around the
induced Meckel's cartilage (Fig.
6C,D, arrow) in the vicinity of the lateral and distal ectoderm
where Bmp4 transcripts (Fig.
6B,B') and a faint expression of Shh
(Fig. 6E, arrow) were also
present.
We were then interested in investigating whether QT6-Shh cell transplants could trigger the development of the nervous and muscular tissues normally associated with the mandible. We looked at specific markers of nerve fibers (HNK1 immunostaining) and muscle cells (MyoD gene expression) in grafted E6 chick embryos. HNK1-positive nerve fibers were detected in the ectopic as well as the normal mandibles. HNK1 labeling was found between Meckel's cartilage anlage (expressing Col2a1) and Bmp4-expressing ectoderm (Fig. 6F, arrows; Fig. 6B',C', outlined). MyoD transcripts were also observed in the supernumerary Md, associated with HNK1-immunostained nerve fibers (Fig. 6G,G', arrow).
These results indicate that, at early stages of chick development (5-8 ss), before NCC emigration, Shh can induce the triplication of lower jaws from the caudal part of BA1. The genes encoding the signaling molecules crucial for the onset of jaw development, such as Bmp4, Fgf8 and Shh first are activated in ectopic positions in the ectoderm. This is accompanied by the expression of transcription factors (such as Pitx1, Dlx5, dHand) in the mesenchyme. These processes involve the triggering of the developmental program for cartilage and bone differentiation from NC-derived mesenchyme and of muscle from somite-derived cells.
Shh does not induce ectopic BA1 skeletal structures in other cephalic regions than presumptive BA1 territory
We have demonstrated so far that Shh can trigger the molecular program for
supernumerary lower-jaw development in BA1 territory, knowing that Meckel's
cartilage could not develop in the absence of Shh, even though the different
elements constituting BA1 (ectoderm, endoderm, mesoderm and NC-derived
mesenchyme) were present (Brito et al.,
2006; Melnick et al.,
2005; Yamagishi et al.,
2006). In a second step, we have addressed the issue of whether
Shh could instruct other cephalic regions to acquire BA1 properties. Thus, we
grafted QT6-Shh cells in the presumptive BA2 at 8-9 ss
(Fig. 7A, circle). Three days
later, at E4, QT6-Shh cells were found in the proximal part of BA2
(Fig. 7D, arrowhead). In
contrast to the BA1 grafts, Bmp4 and Fgf8 expression
patterns were not modified (Fig.
7E,F). Moreover, no ectopic induction of Shh gene
activity in BA2 ectoderm was detected. In fact, a downregulation of the
endogenous Shh expression was observed in BA2 (n=4)
(Fig. 7D, arrows). Moreover,
Pitx1 expression was not induced in BA2 mesenchyme
(Fig. 7G). These experiments
showed that, although QT6-Shh cells are capable of modifying Fgf8,
Bmp4 and Pitx1 expression in BA1, no similar effect occurs in
BA2. Later on, at E11, in most cases (n=4/5), no supernumerary
skeletal structures were detected among BA2 derivatives
(Fig. 7C). Only one embryo
showed a small bifurcation of the basihyal (data not shown). The morphology of
the face and neck was normal except for the larger middle and inner ear
observed on the grafted side (Fig.
7B, arrow). This shows that the patterning effect of Shh on BA1 is
not reproducible in BA2.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The arrangement of the extra jaws that are induced by the QT6-Shh cells is strikingly reproducible: the proximal part of the Meckel's cartilage is common and distal branches grow separately as mirror images.
Shh signal is pivotal for the development of Meckel's cartilage and associated structures
It has previously been shown in our laboratory that a fragment of anterior
ventral foregut endoderm is capable of inducing a supernumerary lower beak
when grafted in BA1 presumptive area of 6 ss chick embryos. Moreover, excision
of the same fragment of endoderm on one side is followed by the absence of the
corresponding Meckel's cartilage (Couly et
al., 2002). In recent work, we showed that after excision of
Shh-expressing anterior foregut endoderm along with the entire
forehead at early neurula stages (5-6 ss), the cephalic NCCs which colonize
BA1 exiting from the mesencephalon migrated normally but were further on
subjected to massive apoptosis, whereas Shh was not expressed in the
pharyngeal endoderm. Fgf8 and Bmp4 transcripts were absent
in BA1 ectoderm at E3-4 and the embryos were further deprived of Meckel's
cartilage and lower jaw (Brito et al.,
2006; Le Douarin et al.,
2007). These data suggested that the ability of the anterior
pharyngeal endoderm to instruct BA1 to form an ectopic lower beak
(Couly et al., 2002) might be
related to its capacity to produce Shh. This prompted us to graft
Shh-producing cells in the presumptive BA1 at 5-8 ss in order to see if these
cells can fully mimic endoderm grafts in this process. It turned out that an
ectopic source of Shh, placed on the right side of the embryo, leads to the
triplication of the lower jaw with mirror-image polarities along the
rostrocaudal axis. It has to be stressed that, in Couly's experiments, the
grafted ventral pharyngeal endoderm exerts a strong effect on the final shape
and position of the ectopic lower jaw: with QT6-Shh cells, the endogenous and
extra Meckel's cartilages are fused on their proximal regions that distally
diverge in three independent branches; by contrast, the endoderm graft leads
to the formation of one ectopic Meckel's cartilage parallel to the endogenous
one. When the ventral pharyngeal endoderm was grafted bilaterally, a complete
extra lower jaw was even induced (Couly et
al., 2002).
In none of the experiments reported here
(Couly et al., 2002;
Brito et al., 2006) (the
present work) was the morphogenesis of the proximal (dorsal) part of the jaw
perturbed. When the forehead was excised together with the PcP and the rostral
Shh-expressing endoderm, no Meckel's cartilages and associated bones
were present but the quadrate and articular cartilages, as well as the
squamosal, developed normally (Brito et
al., 2006). In the experiments by Couly et al.
(Couly et al., 2002), no
duplication of these cartilages occurred. The same is true for the experiments
that we describe here. Thus, Shh is likely to control the formation and
extension of Meckel's cartilage and associated structures, whereas the
formation of the proximal (dorsal) skeleton (quadrate and articular) depends
upon different mechanisms. In spite of the differences in the final shape of
the supernumerary lower beaks obtained with QT6-Shh cells or with foregut
endoderm grafts, Shh alone is sufficient to initiate the lower-jaw
developmental program in the caudal presumptive BA1 territory. Induction of
mirror-image duplication of Meckel's cartilage by Shh-producing cells recalls
the effect of the same type of graft in the anterior mesenchyme of the limb
bud. The latter results in digit duplication with mirror-image orientation,
owing to the induction of a new ZPA
(Riddle et al., 1993).
However, in the limb bud, Shh expression was not induced in the
ectoderm.
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;
Haworth et al., 2007;
Brito et al., 2006). As shown
in our schematic model (Fig.
8), at 6 ss, the pharyngeal endoderm expressing Shh is in
close contact with the ventral ectoderm, thus forming the oro-pharyngeal
membrane (Fig. 8A'),
which further on will form the epithelium lining the oral opening
(Fig. 8A''). At this stage
(E4), expressions of both Fgf8 and Bmp4 genes are activated
in the oral epithelium, which corresponds to the rostral-most BA1 ectoderm.
Moreover, Shh is expressed in the pharyngeal endoderm in close
contact with Fgf8 proximally and Bmp4 distally
(Fig. 8C, part I, on the
right). In our experiments, 7 hours after the graft (at 10 ss), the QT6-Shh
cells are located between the endoderm and ectoderm of the presumptive BA1
(Fig. 8B; see
Fig. 1B). Later on (at E4), the
quail fibroblasts will be found exclusively in the proximal BA1 area
(Fig. 8C', purple dots).
Re-patterning of BA1 ectoderm by QT6-Shh cells involves a cascade of events
similar to those taking place normally in BA1 ectodermal epithelium under the
influence of the foregut endoderm. A new `oral-like epithelium' is induced in
which Fgf8, Bmp4 and Shh are expressed in the caudal part of
BA1 ectoderm (Fig. 8C, left;
Fig. 8C'; see
Fig. 3 and Fig. S3 in the
supplementary material). This rostrocaudal re-patterning is also observed in
the mesenchyme through upregulation of Pitx1, Noggin and
dHand, and reduction or loss of Gli3. This suggests that, at
5-8 ss, the capacity to respond to Shh, far from being restricted to the
region fated to become the oral epithelium (rostral BA1 ectoderm), is in fact
present over the entire BA1 ectoderm. The respective spatial disposition of the ectodermal areas expressing Shh, Bmp4 and Fgf8 is highly significant concerning the rostrocaudal orientation of the future extra jaws induced by QT6-Shh (Fig. 8C: 1-3).
Both Bmp4 and Fgf8 have previously been shown to be crucial for lower-jaw
development by other groups. Conditional knockout of Bmp4 in distal
BA1 ectoderm completely hampered lower-jaw development
(Liu et al., 2005). Other
authors found that application of Bmp2/4-soaked beads in chick BA1 mesenchyme,
at E3, induced Meckel's cartilage duplication
(Barlow and Francis-West, 1997;
Mina et al., 2002). In our
experimental design, prior to lower-jaw duplication, Pitx1
(Lanctôt et al., 1999),
Dlx5 (for a review, see Depew et
al., 2005) and dHand
(Yanagisawa et al., 2003) were
associated with Bmp4 expression and followed by the activation of
genes involved in chondrocytes (Sox9 and Col2a1)
(Mori-Akiyama et al., 2003),
osteoblasts (Cbfa1) (Ducy et al.,
1997) and muscle (MyoD)
(Kablar et al., 1998)
differentiation.
The control of Fgf8 expression by Bmp4 was demonstrated in
Chordin and Noggin knockout mice
(Stottmann et al., 2001). In
these cases, Bmp activities were enhanced while the proximal part of BA1 was
devoid of Fgf8 expression, resulting in the absence of lower jaw. The
same phenotype was obtained by downregulation of Bmp4 expression in
BA1 ectoderm, leading to an increase of Fgf8 territory, which
extended distally (Liu et al.,
2005). Head infection with RCAS-Fgf8 also resulted in
shorter lower-jaw formation (Abzhanov and
Tabin, 2004).
It appears therefore that a crucial equilibrium between the production of
Bmp4 and Fgf8 is necessary for the extension of Meckel's cartilage. Our
experiments, which consist of the administration of exogenous Shh prior to BA1
development, produce extra sources of Bmp4 and Shh without downregulating Fgf8
production. This disposition favors the development of extra jaws. In these
experiments, the presence of exogenous Shh can lead to an increase of NCC
number, and NCCs are known to be able to produce Bmp antagonists such as
Noggin (Stottmann et al.,
2001; Smith and Graham,
2001). We could demonstrate that graft of QT6-Shh cells in BA1
resulted in induction of ectopic Noggin expression, corresponding to
Bmp4 areas (see Fig.
3C, Fig. 4A). Under
these conditions, the level of Bmp4 could be restricted to a rate compatible
with the production of Fgf8 in a quantity appropriate for the formation of the
supernumerary jaws. The fact that infection of chicken Md with
RCAS-Noggin prevents the formation of cartilage and bones supports
this view (Foppiano et al.,
2007).
It is interesting that the graft of QT6-Shh cells results in reduction or
loss of Gli3 expression in BA1. This is in line with the fact that
inactivation of Gli3 in mice leads to upregulation of Fgf8
transcripts in several sites, such as in the apical ectodermal ridge of the
limb bud (Aoto et al., 2002),
and results in polydactylism (Litingtung
et al., 2002; te Welscher et
al., 2002a). A hypothesis to account for the loss of Gli3
after QT6-Shh cell grafts in BA1 is an indirect action through dHand,
which has already been shown to control Gli3 expression in the
posterior limb bud of mouse embryo
(Fernandez-Teran et al., 2000;
te Welscher et al., 2002b).
Moreover, it is known that Shh does not act directly on Gli3 gene
expression but rather is involved in preventing the cleavage of full-length
Gli3 (activator form 190 kDa) to its repressor form (83 kDa)
(Wang et al., 2000).
In our experiments, the striking effect of QT6-Shh cell grafts on BA1 development is the induction of ectodermal foci similar to the normal one acting as organizer of lower-jaw extensions. This process leads to the development of two extra lower jaws owing to the maintenance of a balance between Fgf8 and Bmp4 expression levels.
BA1 ectoderm has a specific capacity to respond to Shh signal
The last aspect of this work was to investigate whether Shh was able of
instructing other cephalic areas to acquire BA1 properties. We demonstrated
that QT6-Shh cell grafts in the presumptive area of BA2 neither induced
ectopic skeletal elements nor triggered Fgf8, Bmp4 and Shh
expression in the ectoderm or Pitx1 in the mesenchyme. This is in
agreement with previous studies in which graft of foregut endoderm or FEZ
ectoderm into BA2 did not trigger the development of BA1 structures
(Couly et al., 2002;
Hu et al., 2003). Moreover,
infection of BA2 ectoderm by RCAS-Shh did not result in ectopic
skeleton development (Haworth et al.,
2007). Indeed, Shh expression was disturbed in the
experimental side of BA2 and no hyperplasia was seen comparable with the one
observed in BA1. We also showed that graft of QT6-Shh cells in the presumptive
post-optic region at 4 ss induced ectopic expression of Pitx1 and
Bmp4, but not of Shh and Fgf8, and no ectopic
cartilages and bones were found (Fig.
7J). Similarly, implanting Shh-soaked beads in chick embryo Mx at
HH15 did produced no phenotype (Lee et
al., 2001). By contrast, graft of fibroblasts infected with
RCAS-Shh, into the naso-frontal prominence of a chick embryo at
HH20-21 induced a supernumerary egg tooth and a cartilage rod derived from the
nasal septum (Hu and Helms,
1999). Although these experiments and the ones described here were
carried out at different stages of development, altogether they show that each
cephalic region has its own properties to respond to Shh.
In conclusion, Shh is a pivotal signal for the development of the lower jaw, including Meckel's cartilage and associated skeletal, muscular and neural structures. These morphological events are preceded by Shh, Fgf8 and Bmp4 induction in BA1 ectodermal epithelium, giving rising to a `new oral-like epithelium'. It is notable that this molecular response to Shh is specific to BA1 and cannot be induced in other cephalic regions. Moreover, Shh-producing endodermal cells of the oral epithelium act in lower-jaw development as a zone of polarizing activity (ZPA) comparable with the ZPA in limb patterning. An extra source of Shh applied at an early developmental stage to the BA1 presumptive territory is able to induce two extra zones of polarizing activity caudal to the normal one.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2311/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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