1 Institut dEmbryologie cellulaire et moléculaire du CNRS et du Collège de France, 49bis, avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne cedex, France
2 Hôpital Robert Debré, 48 boulevard Serurier, 75019 Paris, France
Accepted 12 November 2001
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SUMMARY |
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Key words: Foregut endoderm, Neural crest, Facial skeleton, Hox genes, Quail-chick chimeras
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INTRODUCTION |
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Segmentation of the branchial arches close to the hindbrain into rhombomeres constitutes the first visible step in the patterning process of the facial and hypobranchial structures. The rhombencephalon becomes metamerized into eight rhombomeres (Vaage, 1969; Lumsden and Keynes, 1989
) and an iterated series of endodermal pouches form in the pharynx. Specific Hox genes, as well as others, such as Krox20 (Wilkinson et al., 1989
) and valentino (Moens et al., 1996
), are known to be involved in hindbrain segmentation. The Hox genes expressed in the hindbrain belong to the four first paralogue groups, and the anteriormost limit of Hox gene expression corresponds to the boundary between r1 and r2 (Prince and Lumsden, 1994
) (Fig. 1A). As a general rule, the rhombomeres and the neural crest cells that they produce express the same set of Hox genes, forming a Hox code; this rule holds for the crest cells that colonize BA2, BA3 and BA4 (Hunt et al., 1991
). Thus, two domains can be distinguished in the neural crest cells that are at the origin of the facial and visceral skeleton: a rostral Hox gene non expressing domain (Hox negative), which gives rise to the membrane bones of the neurocranium, the nasal capsule, the maxillary bone and the lower jaw; and a Hox-positive domain, which yields the hyoid cartilages (except the entoglossum). It has been shown (Couly et al., 1998
) that Hox-expressing neural crest cells transposed anteriorly to the Hox gene non-expressing domain fail to differentiate into cartilage and bone. By contrast, the neural crest cells of the Hox-negative domain transplanted posteriorly respond normally to the local cues and participate in the formation of normal hyoid cartilage while maintaining their Hox-negative status.
Evidence that Hox gene expression is involved in patterning the visceral skeleton has come from experiments carried out in the mouse, where some genes of the anterior paralogue groups were either mutated or misexpressed. Thus, in Hoxa2-null mutant mice, the neural crest cells of BA2 behave like their Hox-negative counterpart that populate BA1 and form pieces of the lower jaw skeleton (Rijli et al., 1993; Gendron-Maguire et al., 1993
). Further studies have indicated that in normal development, Hoxa2 acts by inhibiting chondrogenesis in the rostral part of BA2 where no other Hox gene is expressed (Kanzler et al., 1998
). This is in line with the deficit in chondrogenesis observed when Hox-positive neural crest cells are transplanted to a Hox-negative domain (Couly et al., 1998
). These and other results (Schilling and Kimmel, 1994
; Sechrist et al., 1994
) support the notion already put forward on the basis of earlier transplantation experiments (Noden, 1983
) that the neural crest cells themselves possess the intrinsic information required for determining the identity of the various bones and cartilages that form the facial skeleton.
It has to be emphasized, however, that other tissue components, the endoderm, mesoderm and superficial ectoderm, participate in facial and visceral arch morphogenesis and may influence neural crest development. It has been shown that neural crest cells can differentiate into cartilage only in the presence of pharyngeal endoderm, whereas ectoderm is crucial for membrane bone differentiation from ectomesenchymal cells (Le Douarin, 1982; Takahashi et al., 1991
). The paraxial mesoderm, in which a discrete segmentation into somitomeres has been described, exerts an effect on neural crest cell migration and differentiation (Trainor and Krumlauf, 2000
). It has recently been shown that the segmental characteristics of the endodermal pharyngeal pouches develop independently of the presence of neural crest cells (Veicht et al., 1999
). Moreover, endodermal segmentation depends upon the activity of specific genes. For example, in vgo null mutant zebrafish, larval hindbrain segmentation proceeds normally but the endodermal gill slits do not form and the distinct streams of neural crest cells exiting from the rhombencephalon ventrally fuse and do not form the individual skeletal elements of the viscerocranium (Piotrowski and Nusslein-Volhard, 2000
). This suggests that at least part of the information for patterning the face could originate from signals of endodermal origin.
We have further explored the origin of the developmental signals responsible for determining the shape of the individual facial bones, as well as their relative positions. We first established that the neural crest cells that form the bones and the cartilages do not themselves possess all the information necessary for patterning the facial skeleton. We then tested the capacity of the foregut endoderm to specify facial bone identity in the avian neurula by performing ablations or grafts of defined endodermal regions.
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MATERIALS AND METHODS |
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Excision and grafts of the neural fold in the Hox-non expressing domain of the neural crest
Chick embryos were prepared for in ovo surgery as described (Teillet et al., 1998). Concurrently, stage-matched quail embryos were harvested and isolated in sterile PBS. The chick embryos were subjected to the surgical ablation of the bilateral neural folds from the mid-diencephalon down to the presumptive r2/r3 limit (Fig. 2A). In certain experimental series, different fragments of the quail neural fold were transplanted into the chick at the posterior diencephalic level (Fig. 2B-D). The fate map of the cephalic neural primordium established by Grapin-Botton et al. (Grapin-Botton et al., 1995
) (Fig. 1A) served as a reference to determine the presumptive level of the various encephalic structures: anterior and posterior diencephalon and mesencephalon and rhombomeres (r) 1 to 8 (Fig. 1). Chimeric embryos were allowed to grow from embryonic day (E) 4 to E8, depending on the experimental series. In another set of experiments, we ablated unilaterally the diencephalic neural fold, leaving the contralateral side unperturbed. The space resulting from the excision was filled with a fragment of equivalent length taken from the quail neural fold at the diencephalic, posterior mesencephalic or rhombencephalic (r4-r6) level (Fig. 2E-G).
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Endodermal stripes were cleared of paraxial cephalic mesoderm as completely as possible. However, a few adherent mesodermal cells may be transplanted together with the endodermal layer. In previous experiments (Couly et al., 1992) (G. Couly, unpublished) transplantations or excisions of definite regions of cephalic mesoderm in 5-6 ss chick and quail embryos never perturbed morphogenesis of the facial skeleton. We, thus, assumed that the effects observed after foregut manipulations were attributable to the endoderm (or to the endoderm together with the few mesodermal cells that may still adhere to it). The endodermal stripes that are either excised or grafted in these experiments must include the endodermal floor of the foregut. Experiments involving only the dorsal aspect of the foregut had no effect on head morphogenesis.
Manipulations of the foregut endoderm
The endodermal stripes already defined were first subjected to unilateral ablation on the right side of the embryo (Fig. 5A,D, Fig. 6A). Then, the ectoderm was replaced and the embryos were further incubated until E6 to E9 and their skeleton was observed. In some cases, embryos were treated at E5 for in situ hybidization.
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RESULTS |
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When the same experiment was performed, using quail mesencephalic neural folds transplanted to the diencephalic level, the grafted neural crest cells contributed to the same structures as the diencephalic derived crest cells, as observed at E4 (n=3), E5 (n=2), E7 (n=2) and E8 (n=3).
The posterior rhombencephalic (r4-r6) neural fold from 5-6 ss quail implanted at the same diencephalic level into stage-matched chicks yielded neural crest cells that colonized the anterior cephalic area where they contributed to the prosencephalic meninges and pericytes. However, they did not participate in the sclerotic that was exclusively composed of chick host cells originating from the mesencephalon (Fig. 4E-H). These embryos were observed at E4 (n=3) and E5 (n=2). At later stages (E6, n=5; E8, n=8) quail cells were found lining peripheral nerves and forming the connective component of ocular muscles; these quail cells contained Hoxa2 transcripts, showing that they had maintained their original molecular identity while migrating within an environment where Hox genes are not activated (Fig. 4H,K). In the nasal bud, some quail cells formed abnormally dense aggregates but did not participate in the cartilage of the nasal septum that developed, by compensation, from endogenous neural crest of more posterior origin (Fig. 4I-K).
In conclusion, the cartilages and bones of the face originate from the anterior domain of the neural crest in which the genes of the Hox-clusters are not activated. After complete ablation of this domain, the facial skeleton fails to develop, owing to the incapability of the neural crest cells of more posterior origin to replace them. The neural folds of the anterior Hox-negative domain possess, by contrast, a high capacity of regulation: a 150 µm long fragment of the neural fold (corresponding to about a quarter of the total length of the neural folds that normally generate the facial skeleton) is sufficient to ensure the normal development of the head. Under the experimental conditions just described, the territory extending from the diencephalon down to r3 can be considered as an equivalence group in which each fragment of the neural fold is endowed with a virtually similar development potential.
These results indicate that the information for the specific morphogenesis of the facial bones is not carried by the neural crest cells themselves, as previously suggested (Noden, 1983). We then investigated the possible role of the foregut endoderm in this process. In a first step, transverse stripes of foregut endoderm were removed and the consequences of this operation on the facial skeleton were recorded.
Ablation of endodermal stripes of foregut endoderm
Stripe I
Ablation of stripe I was performed on the right side of 14 embryos, of which eight could be recovered at E8. Four were anatomically normal. Four had an abnormal nasal capsule in which the nasal septum was deflected to the left side (not shown). In the next series, stripe I was removed on both sides. This resulted in the strong reduction (or absence) of the nasal bud and later on of the nasal capsule, nasal septum and upper beak (n=5) (Fig. 5A-C).
Stripe II
Ablation of stripe II was performed on 70 embryos (Fig. 5D); 12 survived until E8-E9, in all of which Meckels cartilage was missing in the first branchial arch on the operated side. In one out of 12, part of the articular cartilage was also missing (Fig. 5E,F).
One control embryo was sacrificed at E5 for whole-mount in situ hybridization with the Pitx1 probe. Pitx1 is strongly expressed by the stomodeal ectoderm and the mesenchymal cells of the proximal part of the BA1 at E5 (Fig. 5G). In the embryo subjected to removal of stripe II endoderm on the right side, Pitx1 was normally expressed in the left branchial arch. By contrast, Pitx1 expression was strongly reduced on the operated side while the corresponding BA1 was underdeveloped (Fig. 5H). The remaining small distal zone of Pitx1 expression may correspond to the presence of a distal short piece of Meckels cartilage observed in most of the Alcian Blue stained embryos that were subjected to this operation (see Fig. 5E,F).
Stripe III
Ablation of stripe III was performed on 20 embryos. Five embryos were observed at E9. All were devoid of the articular, the quadrate and the proximal part of Meckels cartilages (Fig. 6A,B). Ablation of stripes II and III together (n=3) resulted in the total absence of the entire skeleton of the first branchial arch (not shown).
Stripe IV
Ablation of stripe IV was performed on 12 embryos; five were observed at E8 and showed a strong reduction of the articular and quadrate cartilages (not shown).
In conclusion, removal of transverse stripes of endoderm from the extreme tip of the foregut (corresponding to the level of the prosencephalon) did not perturb the development of the jaw but affected that of the nasal capsule and upper beak. Ablation of the endoderm of zone II, III and IV, corresponding (respectively) to the levels of the mesencephalon (II, III) and of r1-2 (IV) prevented the morphogenesis of Meckels (zone II and III) and of articular and quadrate cartilages (zone III-IV). Moreover, when cartilaginous bones failed to develop, nearby membrane bones, which normally ossify later directly from the neural crest mesenchyme of the nasofrontal bud or of BA1, did not form either. Thus, in the absence of the nasal septum, the other bones of the nasal capsule are missing. Without the quadrate, articular and Meckels cartilages, the squamosal, pterygoid, quadratojugal, angular, supra-angular, opercular and dentary bones of the jaw did not appear.
The absence of skeletal structures after ablation of defined regions of the endoderm could have been due either to mechanical prevention of neural crest cell migration or to the absence of a specifically localized patterning effect of the endoderm on the ectomesenchyme. To clarify this, we implanted defined regions of 5 ss quail foregut endoderm into the head of stage-matched chicken embryos. In such chimeras, donor cells can be identified immunohistologically with the quail-specific QCPN Mab. In these chimeras, migrating cephalic neural crest cells, exiting from the Hox-negative domain of the encephalic vesicles, are subjected to the influence of ectopic pieces of endoderm, even though the host endoderm is left intact.
Ectopic grafts of defined foregut endodermal areas in the migration pathway of cephalic neural crest cells induce the formation of specific cartilages and bones
Grafts of stripe II quail endoderm were made laterally to the mesencephalon of chicken hosts, thus overlying their endogenous counterpart (Fig. 7A). At E3, the grafts formed vesicles embedded within the host neural crest-derived mesenchyme of BA1 (Fig. 7B). At E5, in situ hybridization showed that Pitx1, usually expressed only within the stomodeal ectoderm and mesenchyme of BA1 (Lanctôt et al., 1997), was reproducibly induced by the stripe II endodermal grafts in an ectopic, subocular position (Fig. 7C, n=3). Consistent with the assumption that BA1 identity is specified in the mesenchyme surrounding the endodermal graft, a supernumerary Meckels cartilage formed near the eye, as seen at E8 (n=2), E9 (n=3) and E12 (n=1). These ectopic cartilages, examined at E6 in two chimeras, developed from host neural crest cells (not shown). The corresponding supernumerary membrane bones of the lower jaw also formed (Fig. 7D).
A bilateral stripe II of quail endoderm was implanted underneath and around endogenous stripes II (Fig. 8A-C) (n=5). An ectopic lower beak was induced in the host (Fig. 8D). It contained not only the two Meckels cartilages but extra lower jaw membrane bones that differentiated in contact to the supernumerary cartilages (Fig. 8E). If the stripe II endodermal fragments were implanted lateral to the endogenous foregut and neural tube [as represented in Fig. 8F,G (n=1)], an extra lower beak with the corresponding skeleton and the two part of entoglossum developed hanging lateral to the lower beak of the host (Fig. 8H,I).
Likewise, a graft of ectopic stripe III endoderm in addition to its host counterpart (Fig. 9A) led to the formation of vesicles of graft origin lateral to the mesencephalon by E3 and the condensation of chick host-origin quadrate cartilage around the graft at E6 (Fig. 9B,C). A supernumerary membranous squamosal bone was associated with the ectopic quadrate at E9 (Fig. 9D,E). Thus, precise regions of the pharyngeal endoderm instruct neural crest cells in BA1 to become the cartilaginous bones of the jaws, and the patterning of adjacent membranous bones. These cartilages are necessary for the subsequent patterning of adjacent membranous bones. The instruction given by ectopic endodermal grafts, placed in the migratory path of BA1 neural crest cells, supersedes that of other local signals for differentiation and patterning.
Facial bone orientation relative to the embryonic axes also depends on an endodermally derived information
When a piece of quail endoderm, including both stripes II and III was implanted above the chicken stripes II and III in its normal AP and mediolateral (ML) orientation (Fig. 10A), a supernumerary lower jaw developed above that of the host, as expected (Fig. 10B,C, Fig. 8). Rostrocaudal inversion of the graft (Fig. 10D) gave rise to a duplicate lower jaw developing toward the back of the head (Fig. 10E,F). Meckels cartilage resulting from a 90º rotation of the endodermal graft to the left (anterior becoming medial, posterior becoming lateral, Fig. 10G), pointed toward the top of the head, at the expense of the ipsilateral eye (Fig. 10H,I). After a 90º rotation of the graft to the right (Fig. 10J), the induced jaw was oriented caudally (Fig. 10K,L). The rostrocaudal position of the early foregut endoderm underlying BA1 therefore translated into the orientation of the distoproximal axis of the jaw.
The foregut endoderm patterning cues influence only the non-Hox-expressing cephalic neural crest cells
As mentioned before, two distinct populations of neural crest cells differentiate into the components of the facial and visceral skull. A rostral, Hox-negative population gives rise to all of the membrane bones of the brain case, face and jaws, as well as the cartilaginous bones of the face; a caudal, Hox-positive population yields the hyoid cartilage (except the entoglossum) (Couly et al., 1996). To test whether the endoderm involved in patterning the Hox-negative neural crest can influence the Hox-expressing, chondrogenic rhombencephalic crest cells, foregut stripes II (Fig. 11A) or III from quail embryo were implanted adjacent to r7-r8 of 5 ss chick recipients. Small nodules of cartilage of chick origin formed around the grafts (n=5) in the host cervical region by E9 (Fig. 11B). However, when the quail anterior mesencephalic neural fold accompanied the endodermal graft (Fig. 11C,E), recognizable cartilages and bones rudiments could be observed at E9. Although reduced in size if compared with their normal endogenous counterpart, Meckels, articular and quadrate cartilages, and some associated membrane bones could be identified at the level of the upper neck (Fig. 11D,F,G; n=3). Note that the shape and size of the skeletal pieces that form in these ectopic locations are as close to their normal counterpart as when the grafts are placed in the facial region (c.f. the experiments described above).
Interestingly, supernumerary bones were strictly of quail origin (not shown) and therefore constructed by the grafted non-Hox-expressing neural crest cells. Supernumerary stripe III endoderm, grafted with a posterior mesencephalic neural fold from the same quail donor, induced quadrate (and squamosal) formation in the neck from donor neural crest cells (Fig. 11E-G). Localized information for constructing specific facial skeletal components hence resides in the anterior pharyngeal endoderm. However, only the equivalence group of cephalic neural crest cells that do not express Hox genes can interpret this information.
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DISCUSSION |
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The anteriormost region of the endoderm, although fated to form Sessels pouch and thus to degenerate, is crucial for specification of the nasal septum and later the nasal capsule, the ethmoid bone, and the upper beak. Stripes of endoderm corresponding to the transverse level of the mesencephalon (stripe II and III) and metencephalon (stripe IV) are necessary for the morphogenesis of the bones derived from the maxillary and mandibular buds forming the first branchial arch (Table 1). The foregut endoderm patterning capacities lie in the lateroventral aspect of the foregut endodermal layer. Thus, the size, shape and orientation of the cartilages forming in the facial anlage of the vertebrate embryo seem to be prefeatured in the endoderm of the early head primordium. Although much progress has been made in the understanding of the mechanisms that underlie cell differentiation, the questions raised by morphogenesis have remained largely unaddressed and the fact, disclosed here, that a definite region of foregut endoderm precisely specifies the shape and orientation (with respect to body coordinates) of every given bone of the face is striking.
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We have also investigated the possible patterning capabilities of the other non neural crest and non endoderm-derived constituents of the branchial arches. By using a similar microsurgical approach, defined fragments of the ectoderm covering the cephalic region of the five-somite stage avian neurula, which correspond to previously defined segments called ectomeres (Couly and Le Douarin, 1990) were either removed or ectopically transplanted. In no case did these experiments perturb the development of the facial skeleton (G. Couly, unpublished). Similar investigations carried out with the cephalic paraxial mesoderm showed that, at these early stages, the various AP regions of the cephalic mesoderm (Couly et al., 1992
) are similarly interchangeable. Thus, in strong contrast to the foregut endoderm, neither the ectoderm nor the mesoderm of the early neurula display patterning properties on the skeletogenic process that leads to the formation of the facial bones (G. Couly, unpublished).
The cells of the Hox-negative domain of the cephalic neural crest behave as an equivalence group
In the Hox-negative domain, the cephalic neural crest cells exhibit a large range of plasticity:
(1) their proliferative capacity can be considerably increased in certain of the experimental situations described above. For example, if a large part (three quarters) of the neural fold that normally participates in head morphogenesis is removed, the remaining segment that is left in situ can regulate the deficiencies caused by the operation and construct the complete skeletal and connective tissue components of a normal head
(2) individual areas can be identified in the diencephalic, mesencephalic and metencephalic neural crest that are normally devoted to participate in a definite set of skeletal structures. Nevertheless this regionalization does not correspond to a strict determination of the cells to a given fate. Limited territories of the anterior Hox-negative neural fold have the capacity to regenerate the whole cephalic neural crest and to yield the entire facial skeleton. Thus, although the anterior domain of the neural crest exhibits a certain degree of specificity in being the only one able to form the facial skeleton, it does not contain the information required to specify each of its constitutive bones and cartilages.
The fact that Hoxa2/ mice crest cells originating from r4 and colonizing BA2 form first arch structures shows that, even in this posterior region, the cephalic neural crest can display the same developmental capabilities as their more anterior counterpart (provided they do not activate Hoxa2). This may also imply that the pharyngeal endoderm of the second branchial pouch is as able, like that of the first one, to specify bones of the lower jaw if it is in contact with Hoxa2-negative neural crest cells. The fate map of the foregut endoderm and the determination of the spatial areas participating in each branchial pouch in the pharynx has not yet been established. The exact contribution of zones II to IV to pharyngeal pouches at later stages, when facial morphogenesis is under way, is not yet known, but work is being carried out to clarify this.
The skeletogenic capacities of neural crest cells depend upon Hox gene expression not only in neural crest cells but also in the other cellular constituents of the branchial arches
The experiments described in this and other articles (Gendron-Maguire et al., 1993; Rijli et al., 1993
; Couly et al., 1998
; Kanzler et al., 1998
; Pasqualetti et al., 2000
) indicate that the effect of Hox-selector genes on the morphogenesis of neural crest derivatives involves cellular interactions between the neural crest cells and the environment within which they differentiate. This is particularly clear for the duplication of the lower jaw, as it can be obtained not only by the inactivation of Hoxa2 in the mouse but also by the anteroposterior transfer of the mesencephalic neural crest to the BA2 level (Noden, 1983
; Couly et al., 1998
). By contrast, if Hox-negative neural crest cells are transferred to the levels of BA3 or BA4, formation of an ectopic lower jaw does not ensue. However, formation of BA1 specific cartilages can be obtained even in this posterior location, provided that Hox-negative neural crest cells are transferred together with this appropriate region of foregut endoderm (see Fig. 11). It is characteristic that BA2 endoderm does not express Hoxa2 or any other Hox gene, whereas that of BA3 and BA4 do. It thus appears that the lower jaw skeleton can develop only if Hoxa2 remains unactivated in both the neural crest cells and their immediate environment. By contrast, forced expression of Hoxa2 in Xenopus embryo induces the homeotic transformation of BA1 into BA2 skeleton (Pasqualetti et al., 2000
). In such a case, not only BA1 neural crest cells but also the surrounding tissues, including the endoderm, do express Hoxa2 from a stage preceding crest cell emigration up to complete facial morphogenesis. This experiment supports the notion that Hoxa2 gene expression is not compatible with facial skeleton development. Similarly, in the chick embryo, homeotic transformation of BA1 into BA2 structures (although at a more discrete level than in Xenopus) could be obtained when the constitutive tissues of BA1 were all subjected to Hoxa2 overexpression. Thus, a bifurcation of tongue skeleton could be seen and was assumed to arise form modified BA1 skeletogenic neural crest cells. Sometimes this extra bone was fused to the remains of Meckels cartilage, thus further supporting the view that it arose as a result of homeosis. By contrast, this did not occur when only the neural crest was targeted before migration. In the latter case, first arch skeletal structures were strongly reduced or absent (Grammatopoulos et al., 2000
). In the experiments that involved the anterior transposition of a Hox-positive (r4-r6) fragment of the neural fold, the neural crest cells were surrounded by an environment where no Hox gene was expressed. Although yielding normal neural derivatives, these neural crest cells were unable to form recognizable cartilage structures. Apoptotic figures observed in certain zones where the grafted neural crest cells were aggregated (S. Creuzet, unpublished) support the contention that, in the absence of an appropriate Hox-positive environment, these cells undergo apoptosis. The homeotic transformations of BA1 into BA2 cartilages, observed in the experiments carried out in Xenopus by Pasqualetti et al. (Pasqualetti et al., 2000
) and on chick by Grammatopoulos et al. (Grammatopoulos et al., 2000
), seem therefore to indicate that overexpression of Hoxa2 exerts, in these circumstances, an antiapoptotic role on the crest-derived chondrogenic precursors.
About the nature and mode of action of the endodermal signal(s) on neural crest cells
The nature of the signal(s) arising from the endoderm is so far unknown but it is interesting to see that the presumptive first arch endoderm (zone II) triggers in the neural crest the expression of Pitx1, a gene normally activated in BA1 ectoderm and neural crest cells during the skeletogenic process leading to lower jaw formation. Moreover, the fact that not only the shape but also the proximodistal and anteroposterior polarity of the skeleton is dictated by the endoderm in our ectopic graft experiments, is in line with the observation of Veitch et al. (Veitch et al., 1999) that the polarity of the branchial pouch endoderm is established in the embryo in the absence of neural crest cells. This was deduced from the observation that the expression of genetic markers such as Bmp7, Fgf8 and Pax1 occurs in the same regions of the pouch endoderm whether neural crest cells immigrate or not. Our experiments show that these polarities are already determined within the foregut endoderm at the early neurula stage, i.e. well before the branchial pouches are formed.
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ACKNOWLEDGMENTS |
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