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Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain
Abstract and Figures
Neural crest cells originate at three discontinuous levels along the rostrocaudal axis of the chick rhombencephalon, centred on rhombomeres 1 and 2, 4 and 6, respectively. These are separated by the odd-numbered rhombomeres r3 and r5 which are depleted of migratory neural crest cells. Here we show elevated levels of apoptosis in the dorsal midline of r3 and r5, immediately following the formation of these rhombomeres at the developmental stage (10-12) when neural crest cells would be expected to emerge at these neuraxial levels. These regions are also marked by their expression of members of the msx family of homeobox genes with msx-2 expression preceding apoptosis in a precisely colocalised pattern. In vitro and in ovo experiments have revealed that r3 and r5 are depleted of neural crest cells by an interaction within the neural epithelium: if isolated or distanced from their normal juxtaposition with even-numbered rhombomeres, both r3 and r5 produce migrating neural crest cells. When r3 or r5 are unconstrained in this way, allowing production of crest, msx-2 expression is concomitantly down regulated. This suggests a correlation between msx-2 and the programming of apoptosis in this system. The hindbrain neural crest is thus produced in discrete streams by mechanisms intrinsic to the neural epithelium. The crest cells that enter the underlying branchial region are organised into streams before they encounter the mesodermal environment lateral to the neural tube. This contrasts sharply with the situation in the trunk where neural crest production is uninterrupted along the neuraxis and the segmental accumulation of neurogenic crest cells is subsequently founded on an alternation of permissive and non-permissive qualities of the local mesodermal environment.
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... Several studies (Anderson and Meier, 1981;Graham et al., 1993;Birgbauer et al., 1995) show that a significantly reduced number of neural crest cells originate from r3. Neural crest emigration from r4 begins at stage 8+ and ceases by stage 11+ (Lumsden et al., 1991). ...
... Post-otic crest (r5,r6,r7) Neural crest cells begin migration at this axial level at stage 9+/10 and cease migration about stage 12 (the exact time has not been reported, to our knowledge). Lineage analysis has demonstrated that, although neural crest cells do originate from r5 (Birgbauer et al., 1995), they do not spread laterally from the neural tube, but instead migrate anteriorly or posteriorly before dispersing laterally at r4 and r6 (Graham et al., 1993;Birgbauer et al., 1995;Shigetani et al., 1995). Extensive FoxD3 expression in the r5, r6 and r7 neural folds is observed by stage 8+ (Fig. 2D), and persists in a population of cells dispersing lateral to the neural tube at r6 and r7 but not r5 at stage 10 ( Fig. 2H; in agreement with Birgbauer et al., 1995). ...
... Extensive FoxD3 expression in the r5, r6 and r7 neural folds is observed by stage 8+ (Fig. 2D), and persists in a population of cells dispersing lateral to the neural tube at r6 and r7 but not r5 at stage 10 ( Fig. 2H; in agreement with Birgbauer et al., 1995). Expression of FoxD3 in the dorsal neural tube is greatly reduced by stage 13, shortly after neural crest cells cease migration at this axial level, and is eliminated altogether in r5, coincident with the time of extensive cell death observed in that rhombomere (Graham et al., 1993). Once migration is under way, this population of the neural crest (also known as the circumpharyngeal crest; Kuratani and Kirby, 1991) spreads laterally and posteriorly to populate branchial arches 3 and 4, and to a limited extent branchial arch 6 (Shigetani et al., 1995;Fig. ...
The winged-helix or forkhead class of transcription factors has been shown to play important roles in cell specification and lineage segregation. We have cloned the chicken homolog of FoxD3, a member of the winged-helix class of transcription factors, and analyzed its expression. Based on its expression in the dorsal neural tube and in all neural crest lineages except the late-emigrating melanoblasts, we predicted that FoxD3 might be important in the segregation of the neural crest lineage from the neural epithelium, and for repressing melanogenesis in early-migrating neural crest cells.
Misexpression of FoxD3 by electroporation in the lateral neural epithelium early in neural crest development produced an expansion of HNK1 immunoreactivity throughout the neural epithelium, although these cells did not undergo an epithelial/mesenchymal transformation. To test whether FoxD3 represses melanogenesis in early migrating neural crest cells, we knocked down expression in cultured neural crest with antisense oligonucleotides and in vivo by treatment with morpholino antisense oligonucleotides. Both experimental approaches resulted in an expansion of the melanoblast lineage, probably at the expense of neuronal and glial lineages. Conversely, persistent expression of FoxD3 in late-migrating neural crest cells using RCAS viruses resulted in the failure of melanoblasts to develop.
We suggest that FoxD3 plays two important roles in neural crest development. First, it is involved in the segregation of the neural crest lineage from the neuroepithelium. Second, it represses melanogenesis, thereby allowing other neural crest derivatives to differentiate during the early stages of neural crest patterning.
... In addition, local foci of cell death also refine the migration of NC cells derived from r3 and r5, which undergo massive apoptosis under the control of adjacent rhombomeres through the production of the factor Bone Morphogenetic Protein 4 (Bmp4). This results in a depletion of mesectodermal cells at the interface between the 1st and 2nd BAs and the 2nd and 3rd BAs, which restricts r4 cell migration to BA2, whose mesenchymal contribution is thus isolated from the adjacent arches [58][59][60]. Together, these mechanisms contribute to the restriction of a cell population in the second arch with a robust molecular identity inherent to its exclusive expression of Homeobox (Hox)a2 gene, whose activity is potentially deleterious for the differentiation of more rostral structures. Furthermore, some molecules can guide CNC cell migration. ...
The neural crest, a unique cell population originating from the primitive neural field, has a multi-systemic and structural contribution to vertebrate development. At the cephalic level, the neural crest generates most of the skeletal tissues encasing the developing forebrain and provides the prosencephalon with functional vasculature and meninges. Over the last decade, we have demonstrated that the cephalic neural crest (CNC) exerts an autonomous and prominent control on the development of the forebrain and sense organs. The present paper reviews the primary mechanisms by which CNC can orchestrate vertebrate encephalization. Demonstrating the role of the CNC as an exogenous source of patterning for the forebrain provides a novel conceptual framework with profound implications for understanding neurodevelopment. From a biomedical standpoint, these data suggest that the spectrum of neurocristopathies is broader than expected and that some neurological disorders may stem from CNC dysfunctions.
... In addition, local foci of cell death also refine the migration of NC cells derived from r3 and r5, which undergo massive apoptosis under the control of adjacent rhombomeres through the production of the factor Bone Morphogenetic Protein 4 (Bmp4). This results in a depletion of mesectodermal cells at the interface between the 1st and 2nd BAs and the 2nd and 3rd BAs, which restricts r4 cell migration to BA2, whose mesenchymal contribution is thus isolated from the adjacent arches [56][57][58]. Together, these mechanisms contribute to the restriction of a cell population in the second arch with a robust molecular identity inherent to its exclusive expression of Hoxa2 gene, whose activity is potentially deleterious for the differentiation of more rostral structures. Furthermore, some molecules can guide CNC cell migration. ...
The neural crest, a unique cell population originating from the primitive neural field, has a multi-systemic and structural contribution to vertebrate development. At the cephalic level, the neural crest generates most of the skeletal tissues encasing the developing forebrain and provides the prosencephalon with functional vasculature and meninges. Over the last decade, we have demonstrated that the cephalic neural crest (CNC) exerts an autonomous and prominent control on forebrain and sense organs development. The present paper reviews the primary mechanisms by which CNC can orchestrate vertebrate encephalization. Demonstrating the role of the CNC as an exogenous source of patterning for the forebrain provides a novel conceptual framework with profound implications for understanding neurodevelopment. From a biomedical standpoint, these data suggest that the spectrum of neurocristopathies is broader than expected and that some neurological disorders may stem from CNC dysfunctions.
... Crest from r4 forms the cartilage elements of the second arch, and crest from r6 and r7 mix to form the cartilages of the third and forth arches. Crest from r3 and r5 undergoes massive apoptosis and so makes a limited contribution to the arches (Graham et al., 1993;Köntges and Lumsden, 1996). ...
Overexpression of Hoxa2 in the chick first branchial arch leads to a transformation of first arch cartilages, such as Meckel’s and the quadrate, into second arch elements, such as the tongue skeleton. These duplicated elements are fused to the original in a similar manner to that seen in the Hoxa2 knockout, where the reverse transformation of second to first arch morphology is observed. This confirms the role of Hoxa2 as a selector gene specifying second arch fate.
When first arch neural crest alone is targeted, first arch elements are lost, but the Hoxa2-expressing crest is unable to develop into second arch elements. This is not due to Hoxa2 preventing differentiation of cartilages. Upregulation of a second arch marker in the first arch, and homeotic transformation of cartilage elements is only produced after global Hoxa2 overexpression in the crest and the surrounding tissue. Thus, although the neural crest appears to contain some patterning information, it needs to read cues from the environment to form a coordinated pattern. Hoxa2 appears to exert its effect during differentiation of the cartilage elements in the branchial arches, rather than during crest migration, implying that pattern is determined quite late in development.
... Rhombomere-specific generation of neural crest (NC) cells is observed along the dorsal part of the hindbrain, resulting in a segmental pathway of migration Sechrist et al., 1993). The even-numbered rhombomeres and r1 generate the vast majority of hindbrain crest cells, whereas r3 and r5 are massively depleted from NC cells through apoptosis (Graham et al., 1993(Graham et al., , 1994, generating small subpopulations that migrate rostrally and caudally into the arches (Sechrist et al., 1993;Köntges and Lumsden, 1996). Hindbrain NC cells migrate ventrally, giving rise to cranial sensory ganglia and populating the pharyngeal arches, thus contributing to the formation of muscular, skeletal and vascular structures (Le Lievre and Le Douarin, 1975;Noden, 1983;Couly et al., 1993;Köntges and Lumsden, 1996). ...
The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3′RARE)/Hoxb1(3′RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3′RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3′RARE) and Hoxa1(null)/Hoxb1(null)double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.
... Normal mouse embryos do exhibit a basal level of dying neural crest cells in their branchial arches that is age-dependent ( Fig. 4; Jeffs et al., 1992;Graham et al., 1993). We therefore compared appropriate developmental stages of α5-null embryos and their wildtype littermates in a statistical study of branchial arch apoptosis. ...
Alpha5beta1 integrin is a cell surface receptor that mediates cell-extracellular matrix adhesions by interacting with fibronectin. Alpha5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the alpha5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of alpha5-null embryos compared to wild-type controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in the alpha5-null embryos. No increases in cell death were seen in alpha5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in alpha5-null embryos, indicating that these subpopulations of cells are dependent on alpha5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh) were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of alpha5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the alpha5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the alpha5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in alpha5-null embryos. These results suggest that alpha5beta1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of mesodermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.
... Moreover, this model was crucial in the analysis of the important morphogenetic processes occurring in the rhombencephalon during embryogenesis. The rhombencephalic neural crest provides most of the mesenchymal cells which are at the origin of the hypobranchial region (see Le Douarin, 1982 for a review; Lumsden et al., 1991;Graham et al, 1993;Sechrist et al., 1993). ...
In this study we have analysed the expression of Hoxb-4, Hoxb-1, Hoxa-3, Hoxb-3, Hoxa-4 and Hoxd-4 in the neural tube of chick and quail embryos after rhombomere (r) heterotopic transplantations within the rhombencephalic area. Grafting experiments were carried out at the 5-somite stage, i.e before rhombomere boundaries are visible. They were preceeded by the establishment of the precise fate map of the rhombencephalon in order to determine the presumptive territory corresponding to each rhombomere. When a rhombomere is transplanted from a caudal to a more rostral position it expresses the same set of Hox genes as in situ. By contrast in many cases, if rhombomeres are transplanted from rostral to caudal their Hox gene expression pattern is modified. They express genes normally activated at the new location of the explant, as evidenced by unilateral grafting. This induction occurs whether transplantation is carried out before or after rhombomere boundary formation. Moreover, the fate of the cells of caudally transplanted rhombomeres is modified: the rhombencephalic nuclei in the graft develop according to the new location as shown for an r5/6 to r8 transplantation.
Transplantation of 5 consecutive rhombomeres (i.e. r2 to r6), to the r8 level leads to the induction of Hoxb-4 in the two posteriormost rhombomeres but not in r2,3,4. Transplantations to more caudal regions (posterior to somite 3) result in some cases in the induction of Hoxb-4 in the whole transplant. Neither the mesoderm lateral to the graft nor the notochord is responsible for the induction. Thus, the inductive signal emanates from the neural tube itself, suggesting that planar signalling and predominance of posterior properties are involved in the patterning of the neural primordium.
The neural crest is a temporary embryonic structure that is composed of a population of multipotent cells that delaminate from the ectoderm by epitheliomesenchymal transformation (► Sect. 5.2). These neural-crest-derived cells or neural crest cells (NCC) contribute to a large number of structures, including the spinal, cranial and autonomic ganglia, the enteric nervous system, the medulla of the adrenal gland, the melanocytes, dermal cells, corneal cells and many of the skeletal and connective tissues of the head (► Sect. 5.3). The whole facial and visceral skeleton and part of the neurocranium are formed from neural crest cells (► Sect. 5.4).A number of craniofacial malformations have major NCC involvement, and are referred to as neurocristopathies (► Sect. 5.5). Under this heading, the oculoauriculo-vertebral spectrum, Treacher Collins syndrome, 22q11.2 deletion syndrome, frontonasal dysplasia, craniosynostoses and CHARGE, Mowat-Wilson and Waardenburg syndromes are discussed. An increasing number of craniofacial disorders is currently classified as neurocristopathies. Many other syndromes might be due to NCC defects and form neurocristopathies. Examples are syndromes caused by environmental factors such as retinoic acid syndrome (► Sect. 5.6), ciliopathies, resulting from defects in primary cilia (► Sect. 5.7) and holoprosencephaly (► Sect. 5.8). Holoprosencephaly is an early disorder of pattern formation that may lead to closely related forebrain and facial malformations. Abnormal development of the skull, caused by craniosynostoses, i.e. craniofacial malformations due to agenesis or premature ossification of the cranial sutures, is discussed in ► Sect. 5.9. Several clinical cases illustrate these disorders.KeywordsNeural crestNeural crest cellsEpitheliomesenchymal transformationCraniofacial malformationsNeurocristopathiesTreacher Collins syndrome22q11.2 deletion syndromeCraniosynostosesHoloprosencephaly
The pharyngeal arches are a series of bulges on the lateral surface of the embryonic head. They are a defining feature of the most conserved, the phylotypic, stage of vertebrate development. In many vertebrate clades, the segmental arrangement of the pharyngeal arches is translated into the iterative anatomy of the gill arches. However, in amniotes the pharyngeal arches undergo a rearrangement during development and the segmental organisation of the pharynx is lost. This remodelling involves the expansion of the second arch which comes to overlie the more posterior arches. A transient sinus forms between the expanded second arch and the posterior arches, that is then lost, and the posterior arches are internalised. The morphogenesis of the second arch has been viewed as being central to this remodelling. Yet little is known about this process. Therefore, in this study, we have characterised the development of the second arch. We show that as the second arch expands, its posterior margin forms a leading edge and that the mesenchymal cells subjacent to this are in an elevated proliferative state. We further show that the posterior marginal epithelium is the site of expression of three key developmental signalling molecules: BMP7, FGF8 and SHH, and that their expression continues throughout the period of expansion. Using a novel approach, we have been able to simultaneously inhibit these three pathways, and we find that when this is done the second arch fails to establish its caudal projection and that there is a loss of proliferation in the posterior mesenchymal cells of the second arch. We have further used this manipulation to ask if the internalisation of the posterior arches is dependent upon the expansion of the second arch. We find that it is not-the posterior arches are still internalised when the expansion of the second arch is curtailed. We further show that while the collapse of the sinus is dependent upon thyroid hormone signalling, that this is not the case for the internalisation of the posterior pouches. Thus, the internalisation of the posterior arches is not dependent on the expansion of the second arch or on the collapse of the sinus. Finally, we show that the termination of expansion of the second arch correlates with a burst of morphogenetic cell death suggesting a mechanism for ending this. Thus, while it has long been thought that it is the morphogenesis of the second arch that drives the remodelling of the pharyngeal arches, we show that this is not the case. Rather the remodelling of the pharyngeal arches is a composite process that can split into contemporaneous but separate events: the expansion of the second arch, the internalisation of the posterior arches and the collapse of the sinus.
Breathing (or respiration) is a complex motor behavior that originates in the brainstem. In minimalistic terms, breathing can be divided into two phases: inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). The neurons that discharge in synchrony with these phases are arranged in three major groups along the brainstem: (i) pontine, (ii) dorsal medullary, and (iii) ventral medullary. These groups are formed by diverse neuron types that coalesce into heterogeneous nuclei or complexes, among which the preBötzinger complex in the ventral medullary group contains cells that generate the respiratory rhythm (Chapter 1). The respiratory rhythm is not rigid, but instead highly adaptable to the physic demands of the organism. In order to generate the appropriate respiratory rhythm, the preBötzinger complex receives direct and indirect chemosensory information from other brainstem respiratory nuclei (Chapter 2) and peripheral organs (Chapter 3). Even though breathing is a hard-wired unconscious behavior, it can be temporarily altered at will by other higher-order brain structures (Chapter 6), and by emotional states (Chapter 7). In this chapter, we focus on the development of brainstem respiratory groups and highlight the cell lineages that contribute to central and peripheral chemoreflexes.
The neural crest cells migrate from their origin into many regions of the vertebrate embryo. Upon reaching their terminal destinations, members of this population undergo cytodifferentiation into a wide range of diverse cell types. To follow the early migratory behavior of avian cephalic neural crest cells, neural fold tissue was transplanted orthotopically from a 3H-thymidine-labeled donor into an unlabeled host. The hosts were sacrificed at subsequent stages and the positions of all labeled neural crest-derived cells ascertained radioautographically. Crest cells emigrating from each of three different preotic regions of the brain displayed unique patterns of migration, which are described in detail.Having established these normal patterns of neural crest cell migration, the following question was posed: Are the unique migratory patterns of the developing neural crest cell population determined within the individual cells prior to their exodus, or are they imposed upon the crest cells by the environment through which the cells move?To resolve this problem neural folds were removed from one of the three cranial regions described above and replaced with a segment of 3H-thymidine-labeled neural fold from either a different cephalic region or the brachial spinal cord level. In nearly every case the heterotopically transplanted cells mimicked the normal patterns of migration, and the embryos were indistinguishable from those which had received orthotopic transplants.This proves that the precise patterns of migration of chick neural crest cells are not irreversibly determined within the cells prior to their emigration. Rather, their complex yet highly organized migratory behavior is largely directed by environmental influences.
1. During the third instar and the prepupal period many degenerating cells are found in the imaginal disks of Calliphora. They can be easily demonstrated in whole disk preparations with the help of any of several basic vital dyes. 2. The degenerating cells become visible ca. 8-10 hours after the onset of cell death and are resorbed within a period of 24 hours. 3. A strong acid phosphatase activity was found to be present in degenerating cells in an early stage of disintegration. 4. The degenerating cells show a very characteristic localization for each disk. The pattern of cell death was described both qualitatively and quantitatively. In the early third instar the localization of the degenerating cells within the pattern is rather variable, but it becomes more constant during development. 5. In the wing disk of the wild type of Drosophila hydei and D. melanogaster a pattern is found that is similar to that in the wing of Calliphora. In so far as it was examined the pattern in the other disks agrees with that of the corresponding disks of Calliphora. 6. In the wing disk of three mutants of Drosophila (beaded, xasta, and vestigial) the pattern of cell death could be related with the missing parts of the adult wing. 7. As judged by its localization the cell death observed probably has a morphogenetic significance. This is discussed with the help of a model.
i. Degenerations of embryonic cells have either been reported as such or have been misinterpreted by various authors as ‘mitotic metabolites’ or blood cells.
2. There is ample support for the morphological identification of dying cells from the following considerations: the degeneration ‘granules’ are initially Feulgen‐positive and have thus originated from nuclear constituents; the stages of cell deaths seen in normal embryos are identical with those produced experimentally and with those observed directly in tissue cultures; degenerating cells react in the same manner to supravital stains in vivo and in vitro.
3. The process of degeneration varies with the degree of specialization of the cell, with its functional state (e.g. mitosis), with the type of animal and under experimental conditions with the causative agents.
4. Cell death may take from less than 1 hr. to about 7 hr. when only a small proportion of a living tissue dies, but may be prolonged to days when numerous cells die simultaneously and their resorption is delayed.
5. Degenerations have been found during the normal development in embryos of all vertebrate animals examined. The occurrence of necrosis in embryos of pure genetical lines is excluded from this article.
6. The incidence of embryonic cell deaths according to site, tissue, developmental stage or process and type of animal is summarized in Table 1.
7. While some degenerations have no obvious function in embryonic development, others seem to play a significant role in embryonic processes, e.g. the morphogenesis and histogenesis of tissues and organs, and the representation and regression of phylogenetic steps (Table 2).
8. Morphogenetic degenerations precede changes in the form of epithelial organs, e.g. during the invagination of the optic cup, the formation of the crystalline lens, the olfactory pit, the neural tube, etc. They bring about the separation of rudiments such as that of the neural tube and the lens from the ectoderm. They reduce the excessive thickening of uniting edges such as those of the body wall and of the mandibles. They are involved in the production of lumina in the solid rudiments of glands and the intestinal tract. In the mesenchyme they precede and make possible the influx of specialized tissue such as the sternal plates or the ingrowth of myogenic tissue in the mandible.
9. Histiogenetic degenerations are related to the differentiation of tissues and organs. The differentiation of the three cell layers of the frog tadpole retina, for instance, is accompanied by three waves of degeneration. Similar cell deaths of early neuroblasts are found in the spinal ganglia outside the limb regions. In amphibia a partial sarcolysis during metamorphosis provides a blastema for the permanent musculature. Sex differentiation of the individual involves the partial degeneration of the Mullerian or Wolffian ducts. Cell deaths also occur in relation to fibre formation and to the appearance of bone and cartilage matrix. Their role in these and in evocatory processes needs further elucidation. Whether cell deaths in the central nervous system and the sense organs at the time of vascularization and neurotization are related to these phenomena remains to be further investigated.
10. Phylogenetic cell deaths are of two types: those which represent a vestigial organ such as the paraphysis or the second muscle stage in higher vertebrates, and those concerned with the regression of larval structures such as the conjunctival papilla, parts of the ganglia of branchial nerves, of the pro‐ and mesonephros. Some of these larval organs have a function in embryonic development, viz. the apical ridge on the limb buds.
11. The causation of the distinctly localized morphogenetic degenerations is obscure. Vascular or nutritional disturbances are unlikely to be responsible for these cell deaths which precede changes in form and appear in the same localizations and amounts in the vascularized tissue of the intact embryo and after explantation in tissue cultures.
. Most of the histiogenetic and phylogenetic cell deaths, as well as some of the not strictly localized morphogenetic degenerations, may be due to the fading out of stimuli for their proliferation or for the completion of their differentiation. If such cells fail to divide, they age and die on reaching the end of their normal life span. This conception assumes that stimuli for the formation of embryonic tissues and organs act for limited periods only and extend over a field of cells. Some of these cells respond fully to stimulation, while others are late to react or do so only partially or receive only a fraction of the whole stimulus. The partial differentiation of cells unfits them for division, for dedifferentiation and redifferentiation in another direction.
12. The localized morphogenetic degenerations are correlated with the incidence and orientation of mitosis and of cell movements, and changes in the form of embryonic organs are brought about by the integration of these three cellular activities. Cell deaths are abundant wherever the regular arrangement and close packing of cells prevent free cell movements; they are rare or absent when, as, for instance, in the tadpole eye, a loose arrangement of cells and a decrease in cell volume (by resorption of yolk) allow of free cell movements.
14. Cell degeneration in vertebrate ontogeny is an important mechanism of integration of cells into tissues and organs by helping to shape the form of organs, by the removal of superfluous cells or by the preparation of a dedifferentiated blastema in histio‐ and phylogenesis.
Here we describe the isolation and characterization of a new chicken homeobox-containing gene, G-Hox 7, which is related to Drosophila msh. The deduced amino acid sequence of the cDNA shows greater than 96% homology to the homeo domain of other vertebrate msh-like genes. As for other species, the amino and carboxy termini of the protein are, however, greatly divergent when compared phylogenetically. In situ hybridization studies revealed the early and widespread expression of G-Hox 7 during chick development. This includes its expression in the primitive streak and extraembryonic cells undergoing epiboly, and its expression along the neural axis, including the forebrain. Expression was also observed in the neural crest, neural crest-derived facial and branchial structures, the otocyst, limb, and heart valves. This widespread and recurrent expression of the transcript suggests that the gene may play an essential role at multiple sites during the initiation of new developmental pathways.
A series of neural crest transplantations has been performed to (1) analyze whether avian premigratory cranial neural crest cells are pluripotential or restricted to specific developmental pathways and (2) examine the ability of trunk neural crest cells to develop in an environment usually occupied by cranial crest cells. Quail embryos, the cells of which have a unique nuclear marker, were used as donors and chick embryos as hosts. Hindbrain crest cells grafted in the place of diencephalic crest cells failed to form neurons in all but one case, in which a small ectopic ganglion was found. In the reciprocal transplants, neural crest cells emigrating from a segment of forebrain crest tissue grafted in the place of metencephalic crest cells produced trigeminal and ciliary ganglia which were completely normal. Thus, crest cells which normally never form ganglionic neurons will do so if placed in a suitable neurogenic environment. These results prove that premigratory avian cranial crest cells are not restricted to specific developmental pathways, but are initially pluripotential. Trunk crest cells grafted in the place of metencephalic crest cells form neuronal ganglia along the proximal trigeminal motor roots but do not form normal trigeminal ganglia. These root ganglia do not display normal peripheral projections, and placode cells, a normal component of the trigeminal ganglion, form ganglia in ectopic locations. Thus, while trunk crest cells respond to the metencephalic environment and form neurons, their response is different from that of cranial crest cells in the same location. Whether this is due to differences in developmental potential or in initial population size is not known.
The differentiation of cephalic neural crest cells into skeletal tissue in birds has been observed using the quail —chick nuclear marking system, which is based on specific differences in the distribution of the nuclear DNA. Chimaeras were formed by replacing a fragment of cephalic neural primordium of a 2- to 12-somite chicken embryo by the corresponding fragment isolated from an equivalent quail embryo. The participation of the graft-derived cells in the formation of the skull of these embryos was studied on histological sections after Feulgen and Rossenbeck staining.
Cells from the pirosencephalic neural crest migrate into the frontal nasal process and mix with the mesencephalic neural crest cells in the lateral nasal processes, around the optic cupule and beneath the diencephalon. In addition, the mesencephalic neural crest cells form the bulk of the mesenchyme of the maxillary processes and mandibular arch, whereas the rhombencephalic neural crest cells become located in the branchial arches.
The origin of cartilages of the chondrocranium and bones of the neurocranium and viscerocranium has been shown in the chimaeric embryos: the basal plate cartilages, occipital bones, sphenoid bones and the cranial vault are mainly of mesodermal origin. However some parts have a dual origin: rhombo-mesencephalic neural crest cells are found in the otic capsule, and the frontal bone, the rostrum of parasphenoid and the orbital cartilages contain diverse amounts of prosencephalo-mesencephalic neural crest cells. The squamosals and the columella auris are formed from mesectodermic cells as are the nasal skeleton, the palatines and the maxillar bones. The mesectodermal origin of mandibular and hyoid bones and cartilages was already known.
From these results it appears that the cephalic neural crest is particularly important in the formation of the facial part of the skull, while the vault and dorsal part are mesodermal and cartilages and bones found in the intermediary region are of mixed origin. The presence of mixed structures implies that the mesoderm and the mesectoderm are equally competent towards the specific inducers of these bones and cartilages. This correlates with the equivalence in differentiation capacities already shown for cephalic mesodeimal and mesectodermal mesenchymes.
The neural crest is the embryonic source of many skeletal and connective tissues associated with the visceral arches of vertebrate embryos. By transplanting quail premigratory neural crest cells, which contain a distinctive nuclear marker [Le Douarin, N. M. (1971). Ann. Embryol. Morphol.4, 125–135], orthotopically into various preotic regions of the chick, it has been shown that all cartilages and bones and most connective tissues of the facial and oral regions are of crest cell origin. These derivatives include the frontal, prefrontal, nasal, maxilla, premaxilla, parasphenoid, palatine, pterygoid, and squamosal bones, in addition to lower jaw skeletal elements. Most connective tissues found in the facial and periprosencephalic regions are of crest origin, including the corneal endothelium, stromal cells, and leptomeningeal tissues. To examine whether crest cells were restricted to specific developmental pathways, quail crest cells from one part of the head were grafted in the place of a different population of chick crest cells. In all cases these host embryos were identical to controls, indicating that cytodifferentiation, growth, and histogenesis of crest-derived tissues are directed by environmental influences encountered by migrating crest cells after they leave their origin.