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Patterning of the neural primordium in the avian embryo
seminars in
CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 157–167
Patterning of the neural primordium in the avian embryo Nicole M. Le Douarin, Anne Grapin-Botton and Martin Catala
induces the floor plate on the ventral midline of the neural plate and of motoneurons on each side of the floor plate.1-3 Since the famous experiments of Otto Mangold in 1933,4 the anteroposterior patterning of the central nervous system (CNS) has generally been considered to result from the effect of qualitatively different neural inducers provided to the neural plate by the mesoderm at different levels of the anteroposterior (AP) axis; each mesodermal level being responsible for the induction of a specific brain or spinal cord structure. Mangold also showed that, once induced, the neural plate itself acquires regionalized inductive capacities by a process designated as homeogenetic induction. The possibility that the specification of the AP neural patterning could result from inductive signals which not only arise from the underlying mesoderm but which may also travel in the plane of the neural epithelium itself was suggested by pioneer experiments carried out in the amphibian embryo by Nieuwkoop,5-7 Nieuwkoop and Nitgevecht8 and EyalGiladi.9 Nieuwkoop5-7 proposed that induction proceeds in two steps. The AP pattern results from the combined effect of an ‘activator’ signal inducing forebrain development and of a ‘transformer’ which modifies the anterior type specification into more posterior ones in a graded manner thus leading to the fine tuning of the AP neural pattern. This second step would be mediated by signals acting within the plane of the neural epithelium. Recent experiments by Doniach10,11 using the Keller-type explants12 in which vertical signalling between ectoderm and mesoderm cannot occur, clearly demonstrated that regionalization of the neuroepithelium can proceed in their absence. It is therefore assumed that this regionalization is established through signals horizontally transmitted within the plane of the ectoderm. In the work that we report here we have addressed two specific questions which pertain to the general problem of the mechanisms through which patterning of the nervous system is established during neurulation in the avian embryo. We have been particularly interested in the development of the tail bud. By using the quail-chick chimera
The quail-chick chimera system has been used to study two problems related to the patterning of the neural primordium. We first report our analysis of the secondary neurulation as it proceeds in the avian tail bud caudally to the posterior neuropore. We show that the territory located caudally to the primary neural tube and joining it to the caudal end of the notochord, designated as the cordoneural hinge (CNH), can be assimilated to the remainder of the Hensen’s node. The CNH undergoes a craniocaudal progression during tail bud development and lays down the notocord and floor plate which thus originate from closely related progenitor cells. We show that the floor plate material becomes inserted within the neurectoderm thus forming the neural plate. The second part of this article is devoted to the hindbrain development. We show that transposition of the presumptive territory of anterior rhombomeres (r1 to r6) at the level of r7–r8 results in their phenotypic posteriorization which is preceded by their expression of posterior Hox genes characteristic to the AP level at which they are transplanted and hence that there are inductive signals within the neural epithelium itself. Key words: hindbrain development/Hox gene expression/ neural development / neural plate / neurulation / quail-chick chimera / rhombomeres ©1996 Academic Press Ltd
IN THE VERTEBRATE EMBRYO, the nervous system arises entirely from the superficial germ layer, the ectoderm. The neural primordium, also called neural plate, appears as a uniform sheet of thickened ectoderm during gastrulation and results from an induction by the underlying axial mesoderm, the notocord. According to this classical view, neural induction and patterning depend on a vertically oriented signaling process taking place as notocordal material folds and invaginates underneath the superficial ectoderm as gastrulation proceeds. Further dorsoventral patterning of the neuroepithelium is considered to involve a second inductive step through which the notocord From Institut d’Embryologie Cellulaire et Mol´eculaire du CNRS et du Coll`ege de France, 49bis, Avenue de la Belle-Gabrielle, 94736 Nogent-sur-Marne Cedex, France ©1996 Academic Press Ltd 1084-9521/96/020157 + 11 $18.00/0
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N. M. Le Douarin et al notochord, the mesenchymal cells of the tail bud generate all the mesodermic structures located beyond the level of the 1st lumbar vertebra. The mechanisms through which the tail bud becomes organized have been the subject of controversies in the past. For Holmdahl13,14 and more recently Griffith et al,17 the tail bud can be assimilated to a regeneration blastema in which the various tissues are formed by apposition of differentiating cells at the caudal end of already formed structures. By contrast, Pasteels15,18 considered the tail bud as a preorganized structure whose development proceeds according to rules similar to those controlling gastrulation. Such a view was demonstrated to be valid in the amphibian embryo.19,20 We thought that the quail-chick marker system could be appropriate to solve this controversy since it would allow the relative movements of the cells forming the tail bud to be demonstrated, thus eventually disclosing a subjacent prepattern and possibly the predetermination of certain territories within this apparently homogenous structure. The experimental design used in this study consisted in the substitution of defined territories of the 25-somite chick tail bud by their counterpart from stage-matched quails and vice versa. The various operations performed are indicated in Figure 2. The graft of region 1 includes the posterior end of the neural tube and notocord plus a zone of undifferentiated cells joining these two structures and designated by Pasteels15 as the cordoneural hinge (CNH). Three types of grafts involving region 1 were done that included either the terminal part of the neural tube only, the neural tube and the dorsal part of the CNH or the entire region 1 with the complete CNH. It should be noted that the endodermal epithelium was included in neither the graft nor the excised territories. Grafts of the terminal part of the neural tube produced the entire spinal cord at the level of sacral nerves 3 to 6 and the corresponding neural crest derivatives.16 When the transplant included the dorsal part of the CNH, the chimeras analysed at E5 and E8 showed that, in addition to the above mentioned neural derivatives, the floor plate of the neural tube located from the level of sacral nerves 6 down to the ventriculus terminalis was formed by quail cells. Finally, when the graft included the totality of the CNH located at the top of the notocord (entire region 1 at the exclusion of the ventral endoderm), all the cells of the notocord and floor plate down to the tail were composed of quail cells (Figure 3A).
system we could follow the progressive organization of the lumbo-sacral and caudal part of the body and perceive the morphogenetic movements that affect the various territories constituting the tail bud. We have thus focused our attention on the formation of the floor plate and notocord during the process designated as ‘secondary neurulation’. From these observations, we have found that the conventional view mentioned above according to which the neural plate is originally a homogeneous structure in which the floor plate is induced by the notocord has to be reconsidered. The second question concerns the mechanisms regulating the establishment of the Hoxcode in the rhombencephalon. We demonstrate that planar inductive signals travelling within the neural epithelium itself are responsible for patterning hindbrain structures via the establishment of the Hox-code in the rhombomeres.
Primary and secondary neurulation: The problem of how the tail bud develops Formation of a neural plate which folds to form a neural tube, a process designated as ‘primary neurulation’, takes place only at the level of the encephalic and cervicotruncal parts of the neural axis. The posterior neuropore closes in the chick and quail embryo at the level of somite 27 which corresponds to the last thoracic vertebra. Caudally to this level, the neural tube forms through a different strategy designated as ‘secondary neurulation’. At the 25-somite stage, the lumbo-sacral and caudal part of the embryo caudal to the posterior neuropore is formed by a mass of undifferentiated cells called tail bud13,14 or ‘bourgeon tronco-caudal’.15 Later on, mesenchymal cells of the tail bud aggregate in the midline, forming a cellular cylinder of densely packed cells. By a transition from a mesenchymal to an epithelial state, the neural tube differentiates through a process called cavitation which involves the formation of a central cavity inside the initially compact neural cylinder. Close to the level of the posterior neuropore, primary and secondary neurulations overlap for a short period and over a short anteroposterior distance (Figure 1). Later on, the secondary neural tube is undistinguishable from its anterior counterpart formed by folding of the preexisting neural plate. Moreover, the notocord segregates ventrally from the median cellular cylinder by formation of a basement membrane separating the floor plate from the notocordal material.16 In addition to the neural tube and the 158
Patterning of the neural primordium in the avian embryo
Figure 1. Histological sections in the avian tail bud at the 25-somite stage (A, sagittal section; B to G, transverse sections, cresyl violet staining). (A) Cranially (right side of the figure), both the neural tube (NT) and notocord (Nc) are individualized whereas the caudal part of the embryo (left side) is composed of the morphologically undifferentiated cells of the tail bud. The chordoneural hinge (CNH) corresponds to a mass of densely packed mesenchymal cells joining the extremities of the notocord and ventral aspect of the neural tube. The ventralmost limit of the tail bud is delimited by the endodermal germ layer (End). (B–G) At the caudalmost end of the tail bud (B), no organized axial structures can be distinguished, but a condensation of mensenchymal cells is visible mediodorsally. More rostrally (C and D), medial cells aggregate to form a solid cord (the so-called medullary cord, MC), in which cavities appear (E) and coalesce to form the lumen of the secondary neural tube (F and G). Scale bars = 100 µm. Reprinted with permission from Catala et al (1995).
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N. M. Le Douarin et al This result shows that, at the 25-somite stage, the caudal part of the already formed neural tube corresponds to the spinal cord of sacral nerve 3 to 6. The CNH itself provides the floorplate–notocord complex which, for a while, forms a single compact structure whose more dorsal part becomes inserted within the neural plate. The neural tube is thus formed through complex morphogenetic movements since its lateral walls and the floorplate joining them have a different origin. The graft of region 2 located caudally to region 1 in the mediodorsal area of the tail bud gives rise to the lateral and dorsal parts of the neural tube (including neural crest derivatives) caudal to the level of sacral nerve 6. As expected, however, the floor plate and notocord are of host origin along the entire posterior axis of the body in these chimeras (observed at E4 and E10; Figure 3B). Although the exchanged territories are located mediodorsally, region 2 also provides the host with somitic cells. The fact that the dermomyotome and the sclerotome are derivated from these cells as well as from chick host cells from the level of the 7th sacral vertebra suggests that region 2 participates, together with an adjacent host’s territory, in the formation of the paraxial sacrocaudal mesoderm. The most rostral limit of the neural tube generated by the graft of region 2, was always more caudal than that of its contribution to the somites. If region 2 is divided into an anterior and posterior moieties, one can see that grafts of the former predominantly yield the
Figure 3. (A) Transverse section of a chimera produced by grafting the entire region 1 of a quail tail bud into a chick embryo at the 25-somite stage. The chimera was analysed at embryonic day 7 on sections treated according to the Feulgen-Rossenbeck’s staining procedure for DNA. The grafted cells give rise to the spinal cord corresponding to the level of sacral nerves 3 to 6. Furthermore, donor cells contribute to the formation of the entire notocord (Nc) and floor plate (arrowheads) from the level of the sixth sacral nerve to the tip of the tail. Scale bar = 10 µm. (B) Transverse section at the level of the last sacral vertebra of a chick embryo at embryonic day 10 stained according to FeulgenRossenbeck reaction in which a quail graft of region 2 was performed at the 25-somite stage. The neural tube is derived from the donor except for the floor plate which is of chick origin. The limit of the floor plate is indicated by arrowheads. Scale bar = 10 µm.
Figure 2. Experiments designed to construct the fate map of the tail bud in the 25-somite embryo. Schematic representation of the avian tail bud at 25-somite stage. (A) Dorsal view. (B) A sagittal section. Each of the territories 1 to 4 were replaced in the chick embryo by their quail counterpart. Regions 1 to 3 are located in the midline from rostral to caudal. Region 1 includes the extremity of the NT and Nc, and the CNH. Region 4 is laterally located. Reprinted with permission from Catala et al (1995).
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Patterning of the neural primordium in the avian embryo precursor cells whose proliferation yields the notocord and ventral part of the neural tube. This sheds a new light on the process of gastrulation in the avian embryo and suggests that a great part of the notocord is formed by the proliferation and extension along the AP axis of material present in the Hensen’s node as the latter undergoes a backward movement. This process, already perceived by Pasteels,18 does not fit with the idea that the notocord is formed by the forward invagination of cells underneath the future neural plate according to the pattern observed in amphibians for the anterior part of the body. Furthermore, the common origin of the notocord and of the floor plate is correlated by the fact that both structures express several genes encoding either transcription factors such as XFKH1/XFD1 and Pintallavis25-27 or morphogenetic substances involved in patterning the neural epithelium (e.g. shh, the vertebrate homologue of the Drosophila gene Hedgehog). It is interesting to notice that in exogastrulas of Xenopus, a certain level of neural induction takes place in the ectoderm by a signal probably emanating from the mesoderm and transmitted horizontally in the plane of the ectoderm. However, the Pintallavis gene is, as expected, expressed in the notocord but not in the midline cells of the neural plate. This has been interpreted as a lack of induction from the notocord,25 but, if the mechanisms of gastrulation disclosed here in the avian embryo can be extended to amphibians, one is led to suggest that the absence of Pintallavis gene expression in the midline of the neural plate in exogastrulas is due to the absence of floorplate cells rather than to the lack of floorplate induction by the notocord.
neural tube whereas the latter essentially contains the somitogenic potencies. Graft of region 3 contributed to the somitic mesoderm together with chick host cells at the same levels as after grafts of region 2. One can infer from this that the somitic mesoderm located caudally to the 6th sacral vertebra is formed by cells located on the mediodorsal region of the tail bud in the 25-somite stage embryo. These cells undergo a migration which leads them first to diverge from the midline area while also expanding along a caudocranial vector. Finally grafts of the lateral mesenchyme indicated as zone 4 label the somites 34 to 36 on the side of the graft. Thus, in this type of chimaeras studied at E6 and E10, the derivatives of the grafted territories are located more cranially than those of regions 2 and 3 and form the totality of the somitic structures corresponding to the level of the 4th to 6th sacral vertebrae. The movement of cells forming the paraxial mesoderm in the developing tail bud are indicated on Figure 4. One can deduce from these studies that tail-bud development corresponds to the continuation of the gastrulation process in which the caudal part of region 2 and region 3 can be assimilated to the remainder of the primitive streak and the CNH to the Hensen’s node. The most important finding in our view is that formation of the sacrocaudal neural tube involves the contribution of two territories spatially separated in the tail bud. One territory is located cranially in the CNH and undergoes an extensive craniocaudal extension to form the floor plate–notocordal complex, whereas another territory is more caudal forming the laterodorsal walls of the neural tube in which the floorplate material will be inserted during formation of the caudal end of the body. These findings are in full agreement with those obtained in Xenopus by Gont et al.20 In frogs, the anterior notocord is produced by the invagination of cells of the marginal zone of the blastoderm.21-23 From stage 13 of Nieuwkoop and Faber,24 however, this process of invagination ends and the notocord is then formed by elongation of the CNH. Gont et al20 showed that cell tracers as well as the expression of the gene Xnot2 located in the CNH at the tail bud stage are found later on in the notocord and in the floor plate. In conclusion, it appears that, at least for the lumbo-sacral and caudal regions of the body, the notocord and floor plate arise together from closely related territories composed of a small population of
Development of the hindbrain: is the Hox-code predetermined in the neuroepithelium prior to rhombomere formation? The rhombencephalon is the only region of the vertebrate central nervous system (CNS) which shows an obvious metamerization during neurogenesis. It is divided into true segmental units called rhombomeres (r) that are transiently visible as undulations of the internal surface of the neuroepithelium.28 The fact that the rhombomeres can be considered as real segments was established on the basis of cellular and molecular data that have accumulated in the last few years. In the chick embryo, vital dye injection into single cells showed that, as soon as the segmentation becomes visible, rhombomeres are characterized by 161
N. M. Le Douarin et al
Figure 4. Fate maps of the avian tail bud established after transplantation of quail grafts as indicated in A. (A) The four transplanted regions represented in a dorsal view of the tail bud at the 25-somite stage. (B,C) Contribution of these regions to the formation of the spinal cord (B, ventral; and C, dorsal views of the spinal cord). The floorplate arises from region 1, whereas the dorsal, lateral and ventro-lateral parts of the neural tube are derived from region 2. (D) Dorsal view of the vertebral column (left side of the figure) and of the spinal cord (right side). Both regions 2 and 3 contribute to the formation of the caudal mesodermal derivatives from the level of somite 37. Region 4 gives rise to somitic derivatives corresponding to the levels of somites 34 to 36. (E) Morphogenetic movements taking place in the tail bud after the 25-somite stage. Mesodermal precursors from region 4 are added to the already formed segmental plate (1). Medial mesodermal cells diverge laterally and migrate rostrally (2). Axial tissues (notochord and neural tube) undergo an elongation allowing their growth in a rostro-caudal direction. LP, lumbar plexus; SP, sacral plexus; PP, pudendal plexus; GB, glycogen body; Sy, synsacrum; FCV, free caudal vertebrae; Py, pygostyle.
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Patterning of the neural primordium in the avian embryo done on mouse and man, several of their avian homologues have now been cloned. We have decided to take advantage of the chick/quail chimera system to see whether Hox gene expression in the rhombencephalon depends on the position of neuroepithelial cells along the AP axis. For this we have performed heterotopic transplantations of the presumptive territory of defined rhombomeres at different levels of the future rhombencephalon between quail and chick embryos. The Hox-code expressed in the quail transplant was then investigated by looking at the expression of the following genes: Hoxa-2, Hoxa3, Hoxb-1, Hoxb-3, Hoxb-4, Hoxd-4 and a-4. The long term fate of the transposed rhombomeres was studied for certain transpositions and compared to that of the same quail rhombomeres grafted isotopically into the chick embryo. It turned out that changing the Hox-code by implanting the neuroepithelium from an anterior to a posterior position profoundly modified their destiny, since the transposed rhombomeres developed according to their novel position along the AP axis and not according to their fate in normal development.38 Most of the experiments were done at the 5-somite stage i.e. before the formation of rhombomere boundaries. In order to know as precisely as possible the anteroposterior limits of the presumptive rhombomeres at these early stages, a prospective map of the rhombencephalic neuroepithelium was established by using DiI and carbone particle marks.
cell lineage restrictions and appear as polyclonal structures between which no or little cell migration takes place.28,29 On the other hand, these units were shown to express some of the homeobox containing (Hox) genes that are considered to be the vertebrate homologues of the homeotic genes that form the HOM-C complex in Drosophila. The vertebrate Hox genes are distributed in four clusters located on four chromosomes in mouse and man (see ref 30 for review). The fact that the genes located at the same position within the different clusters share a high level of homology suggests that vertebrate Hox genes resulted from two consecutive duplications of an ancestor cluster of homeotic genes. As a result of the differential expression of Hox genes along the AP axis, each level is characterized by the expression of a definite set of genes constituting the Hox-code.31 This is particularly so for each pair of rhombomeres in the developing hindbrain. As is most clearly shown for the Hox A and B complexes in the mouse rhombencephalon, the anterior limits of Hox gene expression domains coincide with the limits between two rhombomeres with generally a two segment periodicity. Thus the anterior limit of expression for Hoxb-2 is between r2/r3, for Hoxb-3 between r4/r5 and for Hoxb-4 between r6/r7. Moreover, the rule of colinearity, according to which the Hox genes located on the 3' end of the DNA molecule are those expressed the more cranially and the earliest, is valid in the mouse as it is in the Drosophila embryo. The relationships between the Hox-code and the identity and fate of the rhombomeres were strongly suggested by genetic experiments in which the expression of Hox genes was either suppressed or ectopically driven. For instance, the inactivation of Hoxa-2 resulted in homeotic transformation of second into first arch structures.32,33 The expression of Hoxa-2 in branchial arch 2 seems thus to be necessary for the development of the mesenchymal neural crest derivatives. The inactivation of Hoxa-1 results in the loss or reduction of rhombomeres 4 and 5 and in changes in the fate of neurogenic neural crest cells.34-36 These modifications occur in the rostralmost domain of expression of this gene. When Hoxa-1 is overexpressed throughout the neuroepithelium some neurons derived from r2 acquire a r4 phenotype.37 These results and several other loss-of-function experiments show that Hox genes are necessary for the development of the rhombencephalon and its neurogenic and mesectodermal derivatives. Although most of the work on Hox genes has been
Caudo-cranial transposition of rhombomeres In a first step, the expression of the Hoxa-4, Hoxb-4, Hoxd-4 paralogue genes was tested after unilateral transposition of the presumptive r7/8a (rhombomere 7 and rhombomere 8 facing somite 2) quail territory at the level of r5/6 of a stage-matched chick (5-somite stage). It should be noted that the contralateral unoperated side of the host can be used as control and that this heterospecific combination was justified by the fact that Hox gene expression is similar in the two species. Moreover, the chick Hox probes hybridized similarly to the RNAs of either quail or chick species, and in all cases where the experiments involved chick into chick transplantations, the results were similar to those in which the donor was a quail. In this ectopic position, the transplanted r7/r8a expressed Hoxb-4 as strongly as did the host’s r7 and r8a. Moreover, the contralateral, unoperated side, was negative for Hoxb4 expression. The expression of Hoxa-4 and Hoxd-4 163
N. M. Le Douarin et al epithelium the graft of r5/r6 was placed in r6/7 position. The induction occurred in the graft at the level of r7 but not in r6. Therefore, induction of Hoxb4 respects the normal anterior boundary of expression of this gene which follows the same pattern in the graft and the host. It should be noted that when r5/6 were transplanted into r8 Hoxb-4 paralogues Hoxa-4 and Hoxd-4 were also induced suggesting that the Hox-code was converted from a r5/6 to a r8 code. In r1 to r8 transplantations, Hoxb-4, Hoxa-3 and Hoxa-2 were also induced, showing that the most anterior rhombomeres are also competent for Hox gene induction and that the induction is not restricted to genes of paralogue group 4. These rostral to caudal transpositions reveal that Hox gene expression does not behave in a cell autonomous manner since the grafted rhombomeres express Hoxb-4 according to a pattern consistent with their novel position along the AP axis.
was also maintained in r7/8a transplanted in r5/6. Therefore, at the 5-somite stage, this territory already has the information to express the paralogue genes of rank 4 and their expression was not negatively influenced by surrounding anterior tissues which do not express this gene.
Transposition of anterior rhombomeres to a posterior position: inducibility of Hoxb-4 and other paralogue genes in caudally transplanted rhombomeres In order to see whether Hoxb-4 and other paralogue genes could be induced in territories rostral to their normal limit of expression, r5/6 were transplanted at the r7/8 level. Induction of Hoxb-4 in the transposed r5/6 could be detected as early as at stage 15 of Hamburger and Hamilton,39 corresponding to 16 h after grafting. Interestingly the expression was lower in r7 than in r8 in both the graft and the normal side. When r5/6 was transposed to the posterior region of r8 (r8p), the induction was uniformly distributed on the whole explant as on the contralateral side (Figure 5). In order to see if the anterior limit of expression of Hoxb-4 would be the same in the graft and in the host’s
Origin of the inducing signal Our transplantation studies demonstrate that Hox gene expression patterns and possibly their segmental
Figure 5. Inducibility of Hox genes in the rhombencephalon. (A) r5/6 transplanted unilaterally into r8p. (B) Immunostaining of the quail cells of the graft showing the integration of the quail territory in one side of the chick neuroepithelium. The otic vesicule (OV) indicates the position of endogenous r5/6 of the host. (C) Hoxb-4 is expressed in a gradient from r7 to r8 in the control side. In the grafted side, Hoxb-4 is expressed in a similar gradient in the graft, showing that this gene has been induced in the quail r5/6. The numbers correspond to the host’s rhombomeres.
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Patterning of the neural primordium in the avian embryo the level of r8 downward. In r7, however, where Hoxb-4 is expressed at a lower level than posteriorly, the inductive capacity is probably not sufficient to induce Hoxb-4 expression in the grafted neuroepithelium, even after 2 days.
identity are at least partly determined by adjacent tissues. The above experiments show that the induction does not originate from the neuroepithelium contralateral to the graft through a transversally migrating signal. In contrast a planar signal travelling along the AP axis might be responsible for the induction. Moreover, the lateral or ventral mesoderm, i.e. the notochord might also be responsible for providing the graft with new positional information. Explant-association experiments have previously shown that the anterior notochord is able to induce En-2 expression in the neurectoderm more efficiently than the posterior notocord.40 The notocord might thus be at the origin of the induction of genes containing a homeodomain. In order to test its possible role in reprogramming Hoxb-4 expression in the grafted neural ectoderm, we have implanted a supernumerary notochord fragment from the level of somites 1 to 8 lateroventrally to the presumptive level of r1 to r6 at the 5-somite stage. The presence of the extranotocord originating from the region where Hoxb-4 is strongly expressed in the neural tube did not however induce the activation of this gene in the anterior rhombomeres. At this stage, the notocord also does not seem to be responsible for Hoxb-4 expression in transplanted rhombomeres. To see whether the inductive signals might be transmitted longitudinally in the plane of the neurectoderm, we grafted large rhombomeric territories extending from r2 to r6 (both included) at the level of somites 1 to 4 or 2 to 5 of the host. In these embryos, Hoxb-4 was induced in r5 and r6 (now placed at the level of r8) whereas r2,3,4 remained negative (Figure 6). This result shows that the inducing signal does not come from the tissues lateral to the graft (mesoderm, superficial ectoderm) since Hoxb-4 is inducible in short grafts of r2,3,4 placed in the same position. Therefore, the obvious interpretation of this result is that the inductive signal spreads in the graft along the AP axis from the posterior neural tube, inducing high levels of Hoxb-4 in r6 and lower levels in r5. The induction does not extend significantly when the chimera is observed at E3 (i.e. 2 days after grafting). Although the host’s neuroepithelium expresses Hoxb4 cranially to the graft, the signal in this case does not spread in a rostrocaudal direction since r2–4 remained negative. However, when the r2–6 fragment was grafted more posteriorly, lateral to somites 3–6 or 4–7, Hoxb-4 was induced in r5 and r6 after one day and in the anterior part of the graft after 2 days (Figure 6). This indicates that the inducing substance is present in sufficient amounts to promote the induction from
Correlation of Hox gene induction with phenotypic changes in further differentiation of the rhombomere Since the Hox-code was changed in our grafts, we wondered whether this change in gene-expression pattern resulted in a change in rhombomere identity and further differentiation. We first decided to map
Figure 6. Origin of the inducing signal. (A) When large grafts encompassing r2 to r6 of a quail are transplanted in the chick unilaterally to the level corresponding to r8 and the spinal cord lateral to somites 2–5, Hoxb-4 is induced, but only in the two posterior rhombomeres of the graft. Since the anterior rhombomeres are competent for Hoxb-4 expression, it shows that they do not receive the inductive signal. This signal therefore comes neither from the mesoderm nor from the neuroepithelium lateral to the graft, but is transmitted within the plane of the neuroepithelium and, in A, is present only in the posterior end of the graft. (B) When r2–6 are grafted more posteriorly facing somites 3–6, Hoxb-4 is also induced in the two rostralmost rhombomeres, although after a longer period of time and to a lesser extent in the middle of the graft, showing that at this level the signal can spread from rostral to caudal and perhaps, but less efficiently, from the lateral mesoderm or neuroepithelium.
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N. M. Le Douarin et al experiments described here have been performed in 25-somite stage embryos and therefore concern only the lumbo-sacral and caudal parts of the body. The questions is now raised as to whether formation of more rostral regions of the neural plate proceeds similarly. Experiments are now in progress to answer this question. The quail-chick chimera system was also applied to the problem of hindbrain development. Transposition of rhombomeres at the 5-somite stage between quail and chick embryos demonstrated that Hox gene expression can be induced in anterior rhombomeres transplanted posteriorly along the AP axis. Changing the Hox-code thus resulted in changing the developmental program of the transposed rhombomeres. This experimental design thus allows homeotic transformations to be obtained in the avian embryo and shows that the Hox-code which characterizes rhombomere pairs depends on positional cues. In addition, these experiments demonstrate a strong correlation between the Hox code and the differentiation programme of the rhombomeres. The signal(s) involved in the inductions reported here are still unknown. Retinoic acid (RA) which is distributed in the embryo along a postero-anterior gradient of decreasing concentration is certainly a possible candidate,43 as it has been shown to induce the transcription of Hox genes both in vitro44-46 and in vivo.31,45
the derivatives of rhombomeres 5/6 and 8p. We found that r5/6 motor derivatives were of quail origin for nerves VI, VII and IX–X using homotopic quail-intochick grafts. Moreover, the acoustic nuclei (nucleus laminaris and nucleus magnocellularis) are derived from r5/6 (nucleus magnocellularis extends to r7). The vestibular nucleus nucleus tangentialis is completely derived from r5/6. Derivatives of r5/6 were also found in nucleus pontis medialis and lateralis, nucleus raphe, nucleus olivaris superior, nucleus descendens nervi trigemini and in the vestibular column. Some reticular neurons are also derived from r5/6. r8p grafts (facing somites 3/4) gave rise to motor derivatives in the motor columns of nerves X and XII as well as to the nucleus supraspinalis. This region also participated in the inferior olivary nucleus. In chimeras where quail r5/6 were placed unilaterally in an r8p position in a chick rhombencephalon, the nuclei formed at E841,42 was identical to the contralateral ones of the chick host. The overall shape of the neural tube was similar bilaterally from the rostral to the caudal parts of the graft. The Xth nerve roots were located at the same level on both sides at the rostral end and the XIIth ventral nerve roots at the caudal end of the graft. Nuclei of nerves X and XII, nucleus supraspinalis and both ventral and dorsal inferior olivary nuclei could clearly be distinguished. Moreover, nuclei characteristic of a r5/6 origin (nucleus angularis, tangentialis, laminaris, magnocellularis, abducens) were never observed in the graft.
References
Conclusions
1. Placzeck M, Tessier-Lavigne M, Yamada T, Jessell T, Dodd J (1990) Mesodermal control of neural cell identity: floor plate induction by the notochord. Science 250:985-988 2. Yamada T, Placzek M, Tanaka H, Dodd J, Jessel TM (1991) Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord. Cell 64:635-647 3. Ruiz i Altaba A (1993) Induction and axial patterning of the neural plate: planar and vertical signals. J Neurobiol 24:1276-1304 ¨ 4. Mangold O (1933) Uber die Induktionf¨ahigkeit der verschiedenen Bezirke der Neurula von Urodelen. Naturwiss 21:761-766 5. Nieuwkoop PD (1952a) Activation and organization of the central nervous system in amphibians. III. Synthesis of a new working hypothesis. J Exp Zool 120:83-108 6. Nieuwkoop PD (1952b) Activation and induction of the central nervous system in amphibians. II. Differentiation and organization. J Exp Zool 120:33-81 7. Nieuwkoop PD (1952c) Activation and organization of the central nervous system in amphibians I. Induction and activation. J Exp Zool 120:1-32 8. Nieuwkoop PD, Nitgevecht GV (1954) Neural activation and transformation in explants of competent ectoderm under the influence of fragments of anterior notochord in Urodeles. J Embryol Exp Morph 2:175-193
We have reviewed in this article two sets of results recently obtained concerning the patterning of the early neural primordium in the avian embryo. By constructing quail-chick chimeras we could demonstrate the morphogenetic movements and the changes occurring in the respective positions of embryonic cells during the development of the tail bud. The latter was shown to proceed as the continuation of gastrulation in which the cordoneural hinge (as defined by Pasteels15) is the remain of the Hensen’s node. A similar conclusion was drawn for tail bud development in Xenopus embryo.20 Selective labelling of the CNH by the quail cell marker revealed that it undergoes a rostrocaudal displacement and yields both the notocord and the floor plate. The latter therefore becomes inserted within the future neural ectoderm with which it forms the neural plate as the caudal progression of the CNH proceeds. The 166
Patterning of the neural primordium in the avian embryo 30. Hunt P, Krumlauf R (1991) Deciphering the Hox code:clues to patterning of the branchial region of the head. Cell 66:1075-1078 31. Kessel M, Gruss P (1991) Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67:89-104 32. Gendron-Maguire M, Mallo M, Zhang Z, Gridley T (1993) Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75:1317-1331 33. Rijli FM, Mark M, Lakkaraju S, Dierich A, Doll´e P, Chambon P (1993) A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75:1333-1349 34. Carpenter EM, Goddard JM, Chisaka O, Manley NR, Capecchi MR (1993) Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain. Development 118:1063-1075 35. Mark M, Lufkin T, Vonesch JL, Ruberte E, Olivo J-C, Doll´e P, Gorry P, Lumsden A, Chambon P (1993) Two rhombomeres are altered in Hoxa-1 mutant mice. Development 119:319-338 36. Doll´e P, Lufkin T, Krumlauf R, Mark M, Duboule D, Chambon P (1993) Local alterations of Krox-20 and Hox gene expression in the hindbrain suggest lack of rhombomeres 4 and 5 in homozygote null Hoxa-1 (Hox-1.6) mutant embryos. Proc Natl Acad Sci USA 90:7666-7670 37. Zhang M, Kim H-J, Marshall H, Gendron-Maguire M, Lucas DA, Baron A, Gudas LJ, Gridley T, Krumlauf R, Grippo JF (1994) Ectopic Hoxa-1 induces rhombomere transformation in mouse hindbrain. Development 120:2431-2442 38. Grapin-Botton A, Bonnin M-A, McNaughton LA, Krumlauf R, Le Douarin NM (1995) Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications. Development 121: in press 39. Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49-92 40. Hemmati-Brivanlou A, Stewart RM, Harland RM (1990) Region-specific neural induction of an engrailed protein by anterior notochord in Xenopus. Science 250:800-802 41. Harkmark W (1954) Cell migrations from the rhombic lip to the inferior olive, the nucleus raphe and the pons. A morphological and experimental investigation on chick embryos. J Comp Neurol 100:115-209 42. Tan K, Le Douarin NM (1991) Development of the nuclei and cell migration in the medulla oblongata. Anat Embryol 183:321-343 43. Chen Y, Huang L, Solursh M (1994) A concentration gradient of retino¨ıds in the early Xenopus laevis embryo. Dev Biol 161:70-76 44. Mavilio F, Simeone A, Boncinelli E, Andrews PW (1988) Activation of four homeobox gene clusters in human embryonal carcinoma cells induced to differentiate by retinoic acid. Differentiation 37:73-79 45. Papalopulu N, Lovell-Badge R, Krumlauf R (1991) The expression of murine Hox-2 genes is dependent on the differentiation pathway and displays a collinear sensitivity to retinoic acid in F9 cells and Xenopus embryos. Nucl Acids Res 19:5497-5506 46. Simeone A, Acampora D, Arcioni L, Andrews PW, Boncinelli E, Mavilio F (1990) Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 346:763-766
9. Eyal-Giladi H (1954) Dynamic aspects of neural induction. Arch Biol 65:180-259 10. Doniach T, Phillips CR, Gerhart JC (1992) Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis. Science 257:542-545 11. Doniach T (1993) Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system. J Neurobiol 24:1256-1275 12. Keller RE, Danilchik M (1988) Regional expression, pattern, and timing of convergence and extension during gastrulation of Xenopus laevis. Development 103:193-210 13. Holmdahl DE (1925) Experimentelle Untersuchungen u¨ ber die Lage der Grenze zwischen prim¨arer und sekund¨arer K¨orperentwicklung beim Huhn. Anat Anz 59:393-396 14. Holmdahl DE (1938) Die Morphogenese des Vertebratorganismus vom formalen und experimentellen Gesichtspunkt. Wilhem Roux Arch Entwicklungsmech Org 139:191-226 15. Pasteels J (1937) Etudes sur la gastrulation des vert´ebr´es m´eroblastiques. III. Oiseaux. IV. Conclusions g´en´erales. Arch Biol 48:381-488 16. Catala M, Teillet M-A, Le Douarin NM (1995) Organization and development of the tail bud analyzed with the quail-chick chimaera system. Mech Dev 51:51-65 17. Griffith CM, Wiley MJ, Sanders EJ (1992) The vertebrate tail bud: three germ layers from one tissue. Anat Embryol 185:101-113 18. Pasteels J (1943) Prolif´erations et croissance dans la gastrulation et la formation de la queue des vert´ebr´es. Arch Biol 54:1-51 19. Bijtel JH (1958) The mode of growth of the tail in Urodele larvae. J Embryol Exp Morph 6:466-478 20. Gont LK, Steinbeisser H, Blumberg B, De Robertis EM (1994) Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastoporal lip. Development 119:991-1004 21. Vogt W (1929) Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. II Teil. Gastrulation und Mesodermbildung bei Urodelen und Anuren. Wilhem Roux Arch Entwicklungsmech Org 120:384-706 22. Pasteels J (1942) New observations concerning the maps of presumptive areas of the young amphibian gastrula (Amblystoma and Discoglossus). J Exp Zool 89:255-281 23. Keller RE (1976) Vital dye mapping of the gastrula and neurula of Xenopus laevis. II. Prospective areas and morphogenetic movements of the deep layer. Dev Biol 51:118-137 24. Nieuwkoop PD, Faber J (1967) Normal table of Xenopus laevis. Amsterdam, North Holland 25. Ruiz i Altaba A, Jessel TM (1992) Pintallavis, a gene expressed in the organizer and midline cells of frog embryos: involvement in the development of the neural axis. Development 116:81-93 26. Dirksen ML, Jamrich M (1992) A novel, activin-inducible, blastopore lip specific gene of Xenopus laevis contains a fork-head DNA-binding domain. Genes Dev 6:599-608 27. Kn¨ochel S, Lef J, Clement J, Klocke B, Hille S, Koster M, Kn¨ochel W (1992) Activin-A induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos. Mech Dev 38:157-165 28. Lumsden A (1990) The cellular basis of segmentation in the developing hindbrain. Trends Neurosci 13:329-335 29. Birgbauer E, Fraser S (1994) Violation of cell lineage restriction compartments in the chick hindbrain. Development 120:1347-1356
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Patterning of the neural primordium in the avian embryo Nicole M. Le Douarin, Anne Grapin-Botton and Martin Catala
induces the floor plate on the ventral midline of the neural plate and of motoneurons on each side of the floor plate.1-3 Since the famous experiments of Otto Mangold in 1933,4 the anteroposterior patterning of the central nervous system (CNS) has generally been considered to result from the effect of qualitatively different neural inducers provided to the neural plate by the mesoderm at different levels of the anteroposterior (AP) axis; each mesodermal level being responsible for the induction of a specific brain or spinal cord structure. Mangold also showed that, once induced, the neural plate itself acquires regionalized inductive capacities by a process designated as homeogenetic induction. The possibility that the specification of the AP neural patterning could result from inductive signals which not only arise from the underlying mesoderm but which may also travel in the plane of the neural epithelium itself was suggested by pioneer experiments carried out in the amphibian embryo by Nieuwkoop,5-7 Nieuwkoop and Nitgevecht8 and EyalGiladi.9 Nieuwkoop5-7 proposed that induction proceeds in two steps. The AP pattern results from the combined effect of an ‘activator’ signal inducing forebrain development and of a ‘transformer’ which modifies the anterior type specification into more posterior ones in a graded manner thus leading to the fine tuning of the AP neural pattern. This second step would be mediated by signals acting within the plane of the neural epithelium. Recent experiments by Doniach10,11 using the Keller-type explants12 in which vertical signalling between ectoderm and mesoderm cannot occur, clearly demonstrated that regionalization of the neuroepithelium can proceed in their absence. It is therefore assumed that this regionalization is established through signals horizontally transmitted within the plane of the ectoderm. In the work that we report here we have addressed two specific questions which pertain to the general problem of the mechanisms through which patterning of the nervous system is established during neurulation in the avian embryo. We have been particularly interested in the development of the tail bud. By using the quail-chick chimera
The quail-chick chimera system has been used to study two problems related to the patterning of the neural primordium. We first report our analysis of the secondary neurulation as it proceeds in the avian tail bud caudally to the posterior neuropore. We show that the territory located caudally to the primary neural tube and joining it to the caudal end of the notochord, designated as the cordoneural hinge (CNH), can be assimilated to the remainder of the Hensen’s node. The CNH undergoes a craniocaudal progression during tail bud development and lays down the notocord and floor plate which thus originate from closely related progenitor cells. We show that the floor plate material becomes inserted within the neurectoderm thus forming the neural plate. The second part of this article is devoted to the hindbrain development. We show that transposition of the presumptive territory of anterior rhombomeres (r1 to r6) at the level of r7–r8 results in their phenotypic posteriorization which is preceded by their expression of posterior Hox genes characteristic to the AP level at which they are transplanted and hence that there are inductive signals within the neural epithelium itself. Key words: hindbrain development/Hox gene expression/ neural development / neural plate / neurulation / quail-chick chimera / rhombomeres ©1996 Academic Press Ltd
IN THE VERTEBRATE EMBRYO, the nervous system arises entirely from the superficial germ layer, the ectoderm. The neural primordium, also called neural plate, appears as a uniform sheet of thickened ectoderm during gastrulation and results from an induction by the underlying axial mesoderm, the notocord. According to this classical view, neural induction and patterning depend on a vertically oriented signaling process taking place as notocordal material folds and invaginates underneath the superficial ectoderm as gastrulation proceeds. Further dorsoventral patterning of the neuroepithelium is considered to involve a second inductive step through which the notocord From Institut d’Embryologie Cellulaire et Mol´eculaire du CNRS et du Coll`ege de France, 49bis, Avenue de la Belle-Gabrielle, 94736 Nogent-sur-Marne Cedex, France ©1996 Academic Press Ltd 1084-9521/96/020157 + 11 $18.00/0
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N. M. Le Douarin et al notochord, the mesenchymal cells of the tail bud generate all the mesodermic structures located beyond the level of the 1st lumbar vertebra. The mechanisms through which the tail bud becomes organized have been the subject of controversies in the past. For Holmdahl13,14 and more recently Griffith et al,17 the tail bud can be assimilated to a regeneration blastema in which the various tissues are formed by apposition of differentiating cells at the caudal end of already formed structures. By contrast, Pasteels15,18 considered the tail bud as a preorganized structure whose development proceeds according to rules similar to those controlling gastrulation. Such a view was demonstrated to be valid in the amphibian embryo.19,20 We thought that the quail-chick marker system could be appropriate to solve this controversy since it would allow the relative movements of the cells forming the tail bud to be demonstrated, thus eventually disclosing a subjacent prepattern and possibly the predetermination of certain territories within this apparently homogenous structure. The experimental design used in this study consisted in the substitution of defined territories of the 25-somite chick tail bud by their counterpart from stage-matched quails and vice versa. The various operations performed are indicated in Figure 2. The graft of region 1 includes the posterior end of the neural tube and notocord plus a zone of undifferentiated cells joining these two structures and designated by Pasteels15 as the cordoneural hinge (CNH). Three types of grafts involving region 1 were done that included either the terminal part of the neural tube only, the neural tube and the dorsal part of the CNH or the entire region 1 with the complete CNH. It should be noted that the endodermal epithelium was included in neither the graft nor the excised territories. Grafts of the terminal part of the neural tube produced the entire spinal cord at the level of sacral nerves 3 to 6 and the corresponding neural crest derivatives.16 When the transplant included the dorsal part of the CNH, the chimeras analysed at E5 and E8 showed that, in addition to the above mentioned neural derivatives, the floor plate of the neural tube located from the level of sacral nerves 6 down to the ventriculus terminalis was formed by quail cells. Finally, when the graft included the totality of the CNH located at the top of the notocord (entire region 1 at the exclusion of the ventral endoderm), all the cells of the notocord and floor plate down to the tail were composed of quail cells (Figure 3A).
system we could follow the progressive organization of the lumbo-sacral and caudal part of the body and perceive the morphogenetic movements that affect the various territories constituting the tail bud. We have thus focused our attention on the formation of the floor plate and notocord during the process designated as ‘secondary neurulation’. From these observations, we have found that the conventional view mentioned above according to which the neural plate is originally a homogeneous structure in which the floor plate is induced by the notocord has to be reconsidered. The second question concerns the mechanisms regulating the establishment of the Hoxcode in the rhombencephalon. We demonstrate that planar inductive signals travelling within the neural epithelium itself are responsible for patterning hindbrain structures via the establishment of the Hox-code in the rhombomeres.
Primary and secondary neurulation: The problem of how the tail bud develops Formation of a neural plate which folds to form a neural tube, a process designated as ‘primary neurulation’, takes place only at the level of the encephalic and cervicotruncal parts of the neural axis. The posterior neuropore closes in the chick and quail embryo at the level of somite 27 which corresponds to the last thoracic vertebra. Caudally to this level, the neural tube forms through a different strategy designated as ‘secondary neurulation’. At the 25-somite stage, the lumbo-sacral and caudal part of the embryo caudal to the posterior neuropore is formed by a mass of undifferentiated cells called tail bud13,14 or ‘bourgeon tronco-caudal’.15 Later on, mesenchymal cells of the tail bud aggregate in the midline, forming a cellular cylinder of densely packed cells. By a transition from a mesenchymal to an epithelial state, the neural tube differentiates through a process called cavitation which involves the formation of a central cavity inside the initially compact neural cylinder. Close to the level of the posterior neuropore, primary and secondary neurulations overlap for a short period and over a short anteroposterior distance (Figure 1). Later on, the secondary neural tube is undistinguishable from its anterior counterpart formed by folding of the preexisting neural plate. Moreover, the notocord segregates ventrally from the median cellular cylinder by formation of a basement membrane separating the floor plate from the notocordal material.16 In addition to the neural tube and the 158
Patterning of the neural primordium in the avian embryo
Figure 1. Histological sections in the avian tail bud at the 25-somite stage (A, sagittal section; B to G, transverse sections, cresyl violet staining). (A) Cranially (right side of the figure), both the neural tube (NT) and notocord (Nc) are individualized whereas the caudal part of the embryo (left side) is composed of the morphologically undifferentiated cells of the tail bud. The chordoneural hinge (CNH) corresponds to a mass of densely packed mesenchymal cells joining the extremities of the notocord and ventral aspect of the neural tube. The ventralmost limit of the tail bud is delimited by the endodermal germ layer (End). (B–G) At the caudalmost end of the tail bud (B), no organized axial structures can be distinguished, but a condensation of mensenchymal cells is visible mediodorsally. More rostrally (C and D), medial cells aggregate to form a solid cord (the so-called medullary cord, MC), in which cavities appear (E) and coalesce to form the lumen of the secondary neural tube (F and G). Scale bars = 100 µm. Reprinted with permission from Catala et al (1995).
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N. M. Le Douarin et al This result shows that, at the 25-somite stage, the caudal part of the already formed neural tube corresponds to the spinal cord of sacral nerve 3 to 6. The CNH itself provides the floorplate–notocord complex which, for a while, forms a single compact structure whose more dorsal part becomes inserted within the neural plate. The neural tube is thus formed through complex morphogenetic movements since its lateral walls and the floorplate joining them have a different origin. The graft of region 2 located caudally to region 1 in the mediodorsal area of the tail bud gives rise to the lateral and dorsal parts of the neural tube (including neural crest derivatives) caudal to the level of sacral nerve 6. As expected, however, the floor plate and notocord are of host origin along the entire posterior axis of the body in these chimeras (observed at E4 and E10; Figure 3B). Although the exchanged territories are located mediodorsally, region 2 also provides the host with somitic cells. The fact that the dermomyotome and the sclerotome are derivated from these cells as well as from chick host cells from the level of the 7th sacral vertebra suggests that region 2 participates, together with an adjacent host’s territory, in the formation of the paraxial sacrocaudal mesoderm. The most rostral limit of the neural tube generated by the graft of region 2, was always more caudal than that of its contribution to the somites. If region 2 is divided into an anterior and posterior moieties, one can see that grafts of the former predominantly yield the
Figure 3. (A) Transverse section of a chimera produced by grafting the entire region 1 of a quail tail bud into a chick embryo at the 25-somite stage. The chimera was analysed at embryonic day 7 on sections treated according to the Feulgen-Rossenbeck’s staining procedure for DNA. The grafted cells give rise to the spinal cord corresponding to the level of sacral nerves 3 to 6. Furthermore, donor cells contribute to the formation of the entire notocord (Nc) and floor plate (arrowheads) from the level of the sixth sacral nerve to the tip of the tail. Scale bar = 10 µm. (B) Transverse section at the level of the last sacral vertebra of a chick embryo at embryonic day 10 stained according to FeulgenRossenbeck reaction in which a quail graft of region 2 was performed at the 25-somite stage. The neural tube is derived from the donor except for the floor plate which is of chick origin. The limit of the floor plate is indicated by arrowheads. Scale bar = 10 µm.
Figure 2. Experiments designed to construct the fate map of the tail bud in the 25-somite embryo. Schematic representation of the avian tail bud at 25-somite stage. (A) Dorsal view. (B) A sagittal section. Each of the territories 1 to 4 were replaced in the chick embryo by their quail counterpart. Regions 1 to 3 are located in the midline from rostral to caudal. Region 1 includes the extremity of the NT and Nc, and the CNH. Region 4 is laterally located. Reprinted with permission from Catala et al (1995).
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Patterning of the neural primordium in the avian embryo precursor cells whose proliferation yields the notocord and ventral part of the neural tube. This sheds a new light on the process of gastrulation in the avian embryo and suggests that a great part of the notocord is formed by the proliferation and extension along the AP axis of material present in the Hensen’s node as the latter undergoes a backward movement. This process, already perceived by Pasteels,18 does not fit with the idea that the notocord is formed by the forward invagination of cells underneath the future neural plate according to the pattern observed in amphibians for the anterior part of the body. Furthermore, the common origin of the notocord and of the floor plate is correlated by the fact that both structures express several genes encoding either transcription factors such as XFKH1/XFD1 and Pintallavis25-27 or morphogenetic substances involved in patterning the neural epithelium (e.g. shh, the vertebrate homologue of the Drosophila gene Hedgehog). It is interesting to notice that in exogastrulas of Xenopus, a certain level of neural induction takes place in the ectoderm by a signal probably emanating from the mesoderm and transmitted horizontally in the plane of the ectoderm. However, the Pintallavis gene is, as expected, expressed in the notocord but not in the midline cells of the neural plate. This has been interpreted as a lack of induction from the notocord,25 but, if the mechanisms of gastrulation disclosed here in the avian embryo can be extended to amphibians, one is led to suggest that the absence of Pintallavis gene expression in the midline of the neural plate in exogastrulas is due to the absence of floorplate cells rather than to the lack of floorplate induction by the notocord.
neural tube whereas the latter essentially contains the somitogenic potencies. Graft of region 3 contributed to the somitic mesoderm together with chick host cells at the same levels as after grafts of region 2. One can infer from this that the somitic mesoderm located caudally to the 6th sacral vertebra is formed by cells located on the mediodorsal region of the tail bud in the 25-somite stage embryo. These cells undergo a migration which leads them first to diverge from the midline area while also expanding along a caudocranial vector. Finally grafts of the lateral mesenchyme indicated as zone 4 label the somites 34 to 36 on the side of the graft. Thus, in this type of chimaeras studied at E6 and E10, the derivatives of the grafted territories are located more cranially than those of regions 2 and 3 and form the totality of the somitic structures corresponding to the level of the 4th to 6th sacral vertebrae. The movement of cells forming the paraxial mesoderm in the developing tail bud are indicated on Figure 4. One can deduce from these studies that tail-bud development corresponds to the continuation of the gastrulation process in which the caudal part of region 2 and region 3 can be assimilated to the remainder of the primitive streak and the CNH to the Hensen’s node. The most important finding in our view is that formation of the sacrocaudal neural tube involves the contribution of two territories spatially separated in the tail bud. One territory is located cranially in the CNH and undergoes an extensive craniocaudal extension to form the floor plate–notocordal complex, whereas another territory is more caudal forming the laterodorsal walls of the neural tube in which the floorplate material will be inserted during formation of the caudal end of the body. These findings are in full agreement with those obtained in Xenopus by Gont et al.20 In frogs, the anterior notocord is produced by the invagination of cells of the marginal zone of the blastoderm.21-23 From stage 13 of Nieuwkoop and Faber,24 however, this process of invagination ends and the notocord is then formed by elongation of the CNH. Gont et al20 showed that cell tracers as well as the expression of the gene Xnot2 located in the CNH at the tail bud stage are found later on in the notocord and in the floor plate. In conclusion, it appears that, at least for the lumbo-sacral and caudal regions of the body, the notocord and floor plate arise together from closely related territories composed of a small population of
Development of the hindbrain: is the Hox-code predetermined in the neuroepithelium prior to rhombomere formation? The rhombencephalon is the only region of the vertebrate central nervous system (CNS) which shows an obvious metamerization during neurogenesis. It is divided into true segmental units called rhombomeres (r) that are transiently visible as undulations of the internal surface of the neuroepithelium.28 The fact that the rhombomeres can be considered as real segments was established on the basis of cellular and molecular data that have accumulated in the last few years. In the chick embryo, vital dye injection into single cells showed that, as soon as the segmentation becomes visible, rhombomeres are characterized by 161
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Figure 4. Fate maps of the avian tail bud established after transplantation of quail grafts as indicated in A. (A) The four transplanted regions represented in a dorsal view of the tail bud at the 25-somite stage. (B,C) Contribution of these regions to the formation of the spinal cord (B, ventral; and C, dorsal views of the spinal cord). The floorplate arises from region 1, whereas the dorsal, lateral and ventro-lateral parts of the neural tube are derived from region 2. (D) Dorsal view of the vertebral column (left side of the figure) and of the spinal cord (right side). Both regions 2 and 3 contribute to the formation of the caudal mesodermal derivatives from the level of somite 37. Region 4 gives rise to somitic derivatives corresponding to the levels of somites 34 to 36. (E) Morphogenetic movements taking place in the tail bud after the 25-somite stage. Mesodermal precursors from region 4 are added to the already formed segmental plate (1). Medial mesodermal cells diverge laterally and migrate rostrally (2). Axial tissues (notochord and neural tube) undergo an elongation allowing their growth in a rostro-caudal direction. LP, lumbar plexus; SP, sacral plexus; PP, pudendal plexus; GB, glycogen body; Sy, synsacrum; FCV, free caudal vertebrae; Py, pygostyle.
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Patterning of the neural primordium in the avian embryo done on mouse and man, several of their avian homologues have now been cloned. We have decided to take advantage of the chick/quail chimera system to see whether Hox gene expression in the rhombencephalon depends on the position of neuroepithelial cells along the AP axis. For this we have performed heterotopic transplantations of the presumptive territory of defined rhombomeres at different levels of the future rhombencephalon between quail and chick embryos. The Hox-code expressed in the quail transplant was then investigated by looking at the expression of the following genes: Hoxa-2, Hoxa3, Hoxb-1, Hoxb-3, Hoxb-4, Hoxd-4 and a-4. The long term fate of the transposed rhombomeres was studied for certain transpositions and compared to that of the same quail rhombomeres grafted isotopically into the chick embryo. It turned out that changing the Hox-code by implanting the neuroepithelium from an anterior to a posterior position profoundly modified their destiny, since the transposed rhombomeres developed according to their novel position along the AP axis and not according to their fate in normal development.38 Most of the experiments were done at the 5-somite stage i.e. before the formation of rhombomere boundaries. In order to know as precisely as possible the anteroposterior limits of the presumptive rhombomeres at these early stages, a prospective map of the rhombencephalic neuroepithelium was established by using DiI and carbone particle marks.
cell lineage restrictions and appear as polyclonal structures between which no or little cell migration takes place.28,29 On the other hand, these units were shown to express some of the homeobox containing (Hox) genes that are considered to be the vertebrate homologues of the homeotic genes that form the HOM-C complex in Drosophila. The vertebrate Hox genes are distributed in four clusters located on four chromosomes in mouse and man (see ref 30 for review). The fact that the genes located at the same position within the different clusters share a high level of homology suggests that vertebrate Hox genes resulted from two consecutive duplications of an ancestor cluster of homeotic genes. As a result of the differential expression of Hox genes along the AP axis, each level is characterized by the expression of a definite set of genes constituting the Hox-code.31 This is particularly so for each pair of rhombomeres in the developing hindbrain. As is most clearly shown for the Hox A and B complexes in the mouse rhombencephalon, the anterior limits of Hox gene expression domains coincide with the limits between two rhombomeres with generally a two segment periodicity. Thus the anterior limit of expression for Hoxb-2 is between r2/r3, for Hoxb-3 between r4/r5 and for Hoxb-4 between r6/r7. Moreover, the rule of colinearity, according to which the Hox genes located on the 3' end of the DNA molecule are those expressed the more cranially and the earliest, is valid in the mouse as it is in the Drosophila embryo. The relationships between the Hox-code and the identity and fate of the rhombomeres were strongly suggested by genetic experiments in which the expression of Hox genes was either suppressed or ectopically driven. For instance, the inactivation of Hoxa-2 resulted in homeotic transformation of second into first arch structures.32,33 The expression of Hoxa-2 in branchial arch 2 seems thus to be necessary for the development of the mesenchymal neural crest derivatives. The inactivation of Hoxa-1 results in the loss or reduction of rhombomeres 4 and 5 and in changes in the fate of neurogenic neural crest cells.34-36 These modifications occur in the rostralmost domain of expression of this gene. When Hoxa-1 is overexpressed throughout the neuroepithelium some neurons derived from r2 acquire a r4 phenotype.37 These results and several other loss-of-function experiments show that Hox genes are necessary for the development of the rhombencephalon and its neurogenic and mesectodermal derivatives. Although most of the work on Hox genes has been
Caudo-cranial transposition of rhombomeres In a first step, the expression of the Hoxa-4, Hoxb-4, Hoxd-4 paralogue genes was tested after unilateral transposition of the presumptive r7/8a (rhombomere 7 and rhombomere 8 facing somite 2) quail territory at the level of r5/6 of a stage-matched chick (5-somite stage). It should be noted that the contralateral unoperated side of the host can be used as control and that this heterospecific combination was justified by the fact that Hox gene expression is similar in the two species. Moreover, the chick Hox probes hybridized similarly to the RNAs of either quail or chick species, and in all cases where the experiments involved chick into chick transplantations, the results were similar to those in which the donor was a quail. In this ectopic position, the transplanted r7/r8a expressed Hoxb-4 as strongly as did the host’s r7 and r8a. Moreover, the contralateral, unoperated side, was negative for Hoxb4 expression. The expression of Hoxa-4 and Hoxd-4 163
N. M. Le Douarin et al epithelium the graft of r5/r6 was placed in r6/7 position. The induction occurred in the graft at the level of r7 but not in r6. Therefore, induction of Hoxb4 respects the normal anterior boundary of expression of this gene which follows the same pattern in the graft and the host. It should be noted that when r5/6 were transplanted into r8 Hoxb-4 paralogues Hoxa-4 and Hoxd-4 were also induced suggesting that the Hox-code was converted from a r5/6 to a r8 code. In r1 to r8 transplantations, Hoxb-4, Hoxa-3 and Hoxa-2 were also induced, showing that the most anterior rhombomeres are also competent for Hox gene induction and that the induction is not restricted to genes of paralogue group 4. These rostral to caudal transpositions reveal that Hox gene expression does not behave in a cell autonomous manner since the grafted rhombomeres express Hoxb-4 according to a pattern consistent with their novel position along the AP axis.
was also maintained in r7/8a transplanted in r5/6. Therefore, at the 5-somite stage, this territory already has the information to express the paralogue genes of rank 4 and their expression was not negatively influenced by surrounding anterior tissues which do not express this gene.
Transposition of anterior rhombomeres to a posterior position: inducibility of Hoxb-4 and other paralogue genes in caudally transplanted rhombomeres In order to see whether Hoxb-4 and other paralogue genes could be induced in territories rostral to their normal limit of expression, r5/6 were transplanted at the r7/8 level. Induction of Hoxb-4 in the transposed r5/6 could be detected as early as at stage 15 of Hamburger and Hamilton,39 corresponding to 16 h after grafting. Interestingly the expression was lower in r7 than in r8 in both the graft and the normal side. When r5/6 was transposed to the posterior region of r8 (r8p), the induction was uniformly distributed on the whole explant as on the contralateral side (Figure 5). In order to see if the anterior limit of expression of Hoxb-4 would be the same in the graft and in the host’s
Origin of the inducing signal Our transplantation studies demonstrate that Hox gene expression patterns and possibly their segmental
Figure 5. Inducibility of Hox genes in the rhombencephalon. (A) r5/6 transplanted unilaterally into r8p. (B) Immunostaining of the quail cells of the graft showing the integration of the quail territory in one side of the chick neuroepithelium. The otic vesicule (OV) indicates the position of endogenous r5/6 of the host. (C) Hoxb-4 is expressed in a gradient from r7 to r8 in the control side. In the grafted side, Hoxb-4 is expressed in a similar gradient in the graft, showing that this gene has been induced in the quail r5/6. The numbers correspond to the host’s rhombomeres.
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Patterning of the neural primordium in the avian embryo the level of r8 downward. In r7, however, where Hoxb-4 is expressed at a lower level than posteriorly, the inductive capacity is probably not sufficient to induce Hoxb-4 expression in the grafted neuroepithelium, even after 2 days.
identity are at least partly determined by adjacent tissues. The above experiments show that the induction does not originate from the neuroepithelium contralateral to the graft through a transversally migrating signal. In contrast a planar signal travelling along the AP axis might be responsible for the induction. Moreover, the lateral or ventral mesoderm, i.e. the notochord might also be responsible for providing the graft with new positional information. Explant-association experiments have previously shown that the anterior notochord is able to induce En-2 expression in the neurectoderm more efficiently than the posterior notocord.40 The notocord might thus be at the origin of the induction of genes containing a homeodomain. In order to test its possible role in reprogramming Hoxb-4 expression in the grafted neural ectoderm, we have implanted a supernumerary notochord fragment from the level of somites 1 to 8 lateroventrally to the presumptive level of r1 to r6 at the 5-somite stage. The presence of the extranotocord originating from the region where Hoxb-4 is strongly expressed in the neural tube did not however induce the activation of this gene in the anterior rhombomeres. At this stage, the notocord also does not seem to be responsible for Hoxb-4 expression in transplanted rhombomeres. To see whether the inductive signals might be transmitted longitudinally in the plane of the neurectoderm, we grafted large rhombomeric territories extending from r2 to r6 (both included) at the level of somites 1 to 4 or 2 to 5 of the host. In these embryos, Hoxb-4 was induced in r5 and r6 (now placed at the level of r8) whereas r2,3,4 remained negative (Figure 6). This result shows that the inducing signal does not come from the tissues lateral to the graft (mesoderm, superficial ectoderm) since Hoxb-4 is inducible in short grafts of r2,3,4 placed in the same position. Therefore, the obvious interpretation of this result is that the inductive signal spreads in the graft along the AP axis from the posterior neural tube, inducing high levels of Hoxb-4 in r6 and lower levels in r5. The induction does not extend significantly when the chimera is observed at E3 (i.e. 2 days after grafting). Although the host’s neuroepithelium expresses Hoxb4 cranially to the graft, the signal in this case does not spread in a rostrocaudal direction since r2–4 remained negative. However, when the r2–6 fragment was grafted more posteriorly, lateral to somites 3–6 or 4–7, Hoxb-4 was induced in r5 and r6 after one day and in the anterior part of the graft after 2 days (Figure 6). This indicates that the inducing substance is present in sufficient amounts to promote the induction from
Correlation of Hox gene induction with phenotypic changes in further differentiation of the rhombomere Since the Hox-code was changed in our grafts, we wondered whether this change in gene-expression pattern resulted in a change in rhombomere identity and further differentiation. We first decided to map
Figure 6. Origin of the inducing signal. (A) When large grafts encompassing r2 to r6 of a quail are transplanted in the chick unilaterally to the level corresponding to r8 and the spinal cord lateral to somites 2–5, Hoxb-4 is induced, but only in the two posterior rhombomeres of the graft. Since the anterior rhombomeres are competent for Hoxb-4 expression, it shows that they do not receive the inductive signal. This signal therefore comes neither from the mesoderm nor from the neuroepithelium lateral to the graft, but is transmitted within the plane of the neuroepithelium and, in A, is present only in the posterior end of the graft. (B) When r2–6 are grafted more posteriorly facing somites 3–6, Hoxb-4 is also induced in the two rostralmost rhombomeres, although after a longer period of time and to a lesser extent in the middle of the graft, showing that at this level the signal can spread from rostral to caudal and perhaps, but less efficiently, from the lateral mesoderm or neuroepithelium.
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N. M. Le Douarin et al experiments described here have been performed in 25-somite stage embryos and therefore concern only the lumbo-sacral and caudal parts of the body. The questions is now raised as to whether formation of more rostral regions of the neural plate proceeds similarly. Experiments are now in progress to answer this question. The quail-chick chimera system was also applied to the problem of hindbrain development. Transposition of rhombomeres at the 5-somite stage between quail and chick embryos demonstrated that Hox gene expression can be induced in anterior rhombomeres transplanted posteriorly along the AP axis. Changing the Hox-code thus resulted in changing the developmental program of the transposed rhombomeres. This experimental design thus allows homeotic transformations to be obtained in the avian embryo and shows that the Hox-code which characterizes rhombomere pairs depends on positional cues. In addition, these experiments demonstrate a strong correlation between the Hox code and the differentiation programme of the rhombomeres. The signal(s) involved in the inductions reported here are still unknown. Retinoic acid (RA) which is distributed in the embryo along a postero-anterior gradient of decreasing concentration is certainly a possible candidate,43 as it has been shown to induce the transcription of Hox genes both in vitro44-46 and in vivo.31,45
the derivatives of rhombomeres 5/6 and 8p. We found that r5/6 motor derivatives were of quail origin for nerves VI, VII and IX–X using homotopic quail-intochick grafts. Moreover, the acoustic nuclei (nucleus laminaris and nucleus magnocellularis) are derived from r5/6 (nucleus magnocellularis extends to r7). The vestibular nucleus nucleus tangentialis is completely derived from r5/6. Derivatives of r5/6 were also found in nucleus pontis medialis and lateralis, nucleus raphe, nucleus olivaris superior, nucleus descendens nervi trigemini and in the vestibular column. Some reticular neurons are also derived from r5/6. r8p grafts (facing somites 3/4) gave rise to motor derivatives in the motor columns of nerves X and XII as well as to the nucleus supraspinalis. This region also participated in the inferior olivary nucleus. In chimeras where quail r5/6 were placed unilaterally in an r8p position in a chick rhombencephalon, the nuclei formed at E841,42 was identical to the contralateral ones of the chick host. The overall shape of the neural tube was similar bilaterally from the rostral to the caudal parts of the graft. The Xth nerve roots were located at the same level on both sides at the rostral end and the XIIth ventral nerve roots at the caudal end of the graft. Nuclei of nerves X and XII, nucleus supraspinalis and both ventral and dorsal inferior olivary nuclei could clearly be distinguished. Moreover, nuclei characteristic of a r5/6 origin (nucleus angularis, tangentialis, laminaris, magnocellularis, abducens) were never observed in the graft.
References
Conclusions
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