Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo.

A vital dye analysis of cranial neural crest migration in the chick embryo has provided a positional fate map of greater resolution than has been possible using labelled graft techniques. Focal injections of the fluorescent membrane probe DiI were made into the cranial neural folds at stages between 3 and 16 somites. Groups of neuroepithelial cells, including the premigratory neural crest, were labelled by the vital dye. Analysis of whole-mount embryos after 1-2 days further development, using conventional and intensified video fluorescence microscopy, revealed the pathways of crest cells migrating from mesencephalic and rhombencephalic levels of the neuraxis into the subjacent branchial region. The patterns of crest emergence and emigration correlate with the segmented disposition of the rhombencephalon. Branchial arches 1, 2 and 3 are filled by crest cells migrating from rhombomeres 2, 4 and 6 respectively, in register with the cranial nerve entry/exit points in these segments. The three streams of ventrally migrating cells are separated by alternating regions, rhombomeres 3 and 5, which release no crest cells. Rostrally, rhombomere 1 and the caudal mesencephalon also contribute crest to the first arch, primarily to its upper (maxillary) component. Both r3 and r5 are associated with enhanced levels of cell death amongst cells of the dorsal midline, suggesting that crest may form at these levels but is then eliminated. Organisation of the branchial region is thus related by the dynamic process of neural crest immigration to the intrinsic mechanisms that segment the neuraxis.     

Correlation of HSP110 expression with all-trans retinoic acid-induced apoptosis

In a previous study, we observed the strong expression of a stress protein of the HSP100/Clp family (HSP110) in apoptotic mesectodermal cells during early mouse facial development. In the present study, we describe the strong expression of the same HSP110 in mesectodermal cells undergoing apoptosis after all-trans retinoic acid (RA) administration. We used a teratological model known to increase cell deaths mainly in the first and second branchial arches during mammalian cephalogenesis: the treatment of E9 mouse embryos with all-trans RA, which results in craniofacial malformations comparable to those that characterize mandibulofacial dysostosis in man. Pregnant NMRI mice were treated with 60 mg/kg body weight of all-trans RA, given orally on day 9 of gestation; embryos were taken 4, 12 or 24 hr after RA administration. The apoptotic pattern of RA-induced cell deaths was confirmed using the dUTP biotin nick-end labeling (TUNEL) method and transmission electron microscopy (TEM). HSP110 expression was detected using an immunohistochemical approach. The increase in the number of TUNEL-positive cells and HSP110-positive cells after all-trans RA administration was quantified in the first branchial arch using a computerized method. Twelve hours after RA administration, the increase in the number of HSP110-positive cells is greater than the increase in the number of TUNEL-positive cells. Twenty-four hours after RA administration, only TUNEL-positive cells remain strong in number. We suggest that HSP110 expression could represent a biochemical event of apoptotic cell death induced by RA, associated with early stages of the apoptotic process. In order to find out if HSP110 expression resulted from neosynthesis, we performed in situ hybridization, which demonstrated that the expression of HSP110 occurred at the level of mRNA.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

Head morphogenesis in embryonic avian chimeras: evidence for a segmental pattern in the ectoderm corresponding to the neuromeres

Areas of the superficial cephalic ectoderm, including or excluding the neural fold at the same level, were surgically removed from 3-somite chick embryos and replaced by their counterparts excised from a quail embryo at the same developmental stage. Strips of ectoderm corresponding to the presumptive branchial arches were delineated, thus defining anteroposterior 'segments' (designated here as 'ectomeres') that coincided with the spatial distribution of neural crest cells arising from the adjacent levels of the neural fold. This discrete ectodermal metamerisation parallels the segmentation of the hindbrain into rhombomeres. It seems, therefore, that not only is the neural crest patterned according to its rhombomeric origin but that the superficial ectoderm covering the branchial arches may be part of a larger developmental unit that includes the entire neurectoderm, i.e., the neural tube and the neural crest.     

The role of cell mixing in branchial arch development

Compartmental structures are the basis of a number of developing systems, including parts of the vertebrate head. One of the characteristics of a series of compartments is that mixing between cells in adjacent units is restricted. This is a consequence of differential chemoaffinity between neighbouring cells in adjacent compartments. We set out to determine whether mesenchymal cells in the branchial arches and their precursors show cell-mixing properties consistent with a compartmental organisation. In chimaeric avian embryos we found no evidence of preferential association or segregation of neural crest cells when surrounded by cells derived from a different axial level. In reassociation assays using mesenchymal cells isolated from chick branchial arches at stage 18, cells reformed into clusters without exhibiting a preferential affinity for cells derived from the same branchial arch. We find no evidence for differential chemoaffinity in vivo or in vitro between mesenchymal cells in different branchial arches. Our findings suggest that branchial arch mesenchyme is not organised into a series of compartments.     

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut. This possibility was supported by the observation of neurofilament immunoreactivity in a subset of cells of the premigratory and early migratory neural crest in the vagal, but not other, regions of the neuraxis prior to the appearance of neurofilament immunoreactivity in the branchial arches. Proliferative expansion of cells with neurofilament immunoreactivity was indicated by the observation of mitotic figures in them. It is suggested that the vagal neural crest cells that populate the ENS are already committed to the neuronal lineage while still in the vagal region of the neuraxis. It is therefore not likely that the enteric microenvironment plays a role in this process.     

In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick

The neural crest provides an excellent model system to study invasive cell migration, however it is still unclear how molecular mechanisms direct cells to precise targets in a programmed manner. We investigate the role of a potential guidance factor, neuropilin-1, and use functional knockdown assays, tissue transplantation and in vivo confocal time-lapse imaging to analyze changes in chick cranial neural crest cell migratory patterns. When neuropilin-1 function is knocked down in ovo, neural crest cells fail to fully invade the branchial arches, especially the 2nd branchial arch. Time-lapse imaging shows that neuropilin-1 siRNA transfected neural crest cells stop and collapse filopodia at the 2nd branchial arch entrances, but do not die. This phenotype is cell autonomous. To test the influence of population pressure and local environmental cues in driving neural crest cells to the branchial arches, we isochronically transplanted small subpopulations of DiI-labeled neural crest cells into host embryos ablated of neighboring, premigratory neural crest cells. Time-lapse confocal analysis reveals that the transplanted cells migrate in narrow, directed streams. Interestingly, with the reduction of neuropilin-1 function, neural crest cells still form segmental migratory streams, suggesting that initial neural crest cell migration and invasion of the branchial arches are separable processes.     

The cardiac neural crest in Ambystoma mexicanum.

To establish whether a region of the cranial neural crest contributes cells to the developing heart of Ambystoma mexicanum (axolotl), as it does in many other vertebrates, we constructed a fate map for the neural crest in late neurula stage (stage 19-20) embryos. The fluorescent vital dye, Dil, was used as the lineage label. The various regions of the cranial neural folds were identified in relation to such landmarks as the developing forebrain, midbrain and hindbrain, and the appearance and extent of emerging somites. Labelled cells originating in the rhombencephalic region were found in the aortic arches and in the truncus arteriosus, and occasionally in the walls of the conus arteriosus. Cells were also found in the third and fourth branchial arches. Labelled neural crest from the adjacent anterior trunk region appeared neither in the heart nor the visceral skeleton, whereas those from the mesencephalic region contributed to the first hypobranchial cartilage and to the first three branchial arches, but not to the heart. No labelled cells from any of the regions were seen in the ventricle or auricle.     

Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues

The morphology of skeletal tissues formed in each of the branchial arches of higher vertebrates is unique. In addition to these structures, which are derived from the neural crest, the crest-derived connective tissues and mesodermal muscles also form different patterns in each of the branchial arches. The objective of this study was to examine how these patterns arise during avian embryonic development. Presumptive second or third arch neural crest cells were excised from chick hosts and replaced with presumptive first arch crest cells. Both quail and chick embryos were used as donors; orthotopic crest grafts were performed as controls. Following heterotopic transplantation, the hosts developed several unexpected anomalies. Externally they were characterized by the appearance of ectopic, beak-like projections from the ventrolateral surface of the neck and also by the formation of supernumerary external auditory depressions located immediately caudal to the normal external ear. Internally, the grafted cells migrated in accordance with normal, second arch pathways but then formed a complete, duplicate first arch skeletal system in their new location. Squamosal, quadrate, pterygoid, Meckel's, and angular elements were present in most cases. In addition, anomalous first arch-type muscles were found associated with the ectopic skeletal tissues in the second arch. These results indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural crest population prior to its emigration from the neural epithelium, and not within the pharynx or pharyngeal pouches as had previously been suggested. Furthermore, the patterns of myogenesis by mesenchymal populations derived from paraxial mesoderm is dependent upon properties inherent to the neural crest.     

Transforming growth factor-beta5 expression during early development of Xenopus laevis

Members of the transforming growth factor-beta (TGF-beta) superfamily play various roles during development in both vertebrates and invertebrates. Two isoforms, TGF-beta2 and -beta5, have been isolated from Xenopus laevis. We describe here the localization of TGF-beta5 mRNA in early embryos of X. laevis, assessed by whole-mount in situ hybridization. The first detectable expression of TGF-beta5 was seen in the stage 14 embryo at the posterior tip of notochord, which continued to later stages, accompanied by the expression in bilateral regions of posterior wall in the tail region next to the notochord. At later stages, transient expression was seen in the cement gland (around stage 21) and in the somites (stages 24-27). In addition, expression was present in the branchial arches (stage 29-36) and olfactory placodes (stage 36).     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpress     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

Initial migration and distribution of the cardiac neural crest in the avian embryo: an introduction to the concept of the circumpharyngeal crest

The distribution and migration of the cardiac neural crest was studied in chick embryos from stages 11 to 17 that were immunochemically stained in whole-mount and sectioned specimens with a monoclonal antibody, HNK-1. The following results were obtained: 1) The first phase of the migration in the cardiac crest follows the dorsolateral pathway beneath the ectoderm. 2) In the first site of arrest, the cardiac crest forms a longitudinal mass of neural-crest cells, called in the present study, the circumpharyngeal crest; this mass is located dorsolateral to the dorsal edge of the pericardium (pericardial dorsal horn) where splanchnic and somatic lateral mesoderm meet. 3) A distinctive strand of neural-crest cells, called the anterior tract, arises from the mid-otic level and ends in the circumpharyngeal crest. 4) By stage 16, after the degeneration of the first somite, another strand of neural-crest cells, called the posterior tract, appears dorsal to the circumpharyngeal crest. It forms an arch-like pathway along the anterior border of the second somite. 5) The seeding of the pharyngeal ectomesenchyme takes place before the formation of pharyngeal arches in the postotic area, i.e., the crest cells are seeded into the lateral body wall ventrally from the circumpharyngeal crest; and, by the ventral-ward regression of the pericardial dorsal horn, lateral expansion of pharyngeal pouch, and caudal regression of the pericardium, the crest cell population is pushed away by the pharyngeal pouch. Thus the pharyngeal arch ectomesenchyme is segregated. 6) By stage 14, at the occipital somite level, ventrolateral migration of the neural crest is observed within the anterior half of each somite. Some of these crest cells are continuous with the caudal portion of the circumpharyngeal crest. An early contribution to the enteric neuroblasts is apparent in this area.     

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.     

Experimental study of the origin of the parathyroid glands.

The origin of the parathyroid glands was investigated in chick embryos (Gallus domesticus). Pieces of the third branchial arch were grafted, and its ectodermal layer formed a new structure (parathyroid III), which became separated from the placodial ectoderm. This structure continued to develop until, together with neural crest cells which gave rise to the mesenchyme, it formed a distinct parathyroid III gland by stage 28 of Hamburger and Hamilton.