Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

Cell lineage analysis in neural crest ontogeny

The neural crest is a transitory and pluripotent structure of the vertebrate embryo composed of cells endowed with developmentally regulated migratory properties. We review here a series of studies carried out both in vivo and in vitro on the ontogeny of the neural crest in the avian embryo. Through in vivo studies we established the fate map of the neural crest along the neuraxis prior to the onset of the migration and we demonstrated the crucial role played by the tissue environment in which the crest cells migrate in determining their fate. Moreover, the pathways of neural crest cell migration could also be traced by the quail-chick marker system and the use of the HNK1/NC1 monoclonal antibody (Mab). A large series of clonal cultures of isolated neural crest cells showed that, at migration time, most crest cells are pluripotent. Some, however, are already committed to a particular pathway of differentiation. The differentiation capacities of the pluripotent progenitors are highly variable from one to the other cell. Rare totipotent progenitors able to give rise to representatives of all the phenotypes (neuronal, glial, melanocytic, and mesectodermal) encountered in neural crest derivatives were also found. As a whole we propose a model according to which totipotent neural crest cells become progressively restricted (according to a stochastic rather than a sequentially ordered mechanism) in their potentialities, while they actively divide during the migration process. At the sites of gangliogenesis, selective forces allow only certain crest cells potentialities to be expressed in each type of peripheral nervous system (PNS) ganglia. © 1993 John Wiley & Sons, Inc.     

A subpopulation of cultured avian neural crest cells has transient neurogenic potential

Neural crest cells of vertebrate embryos produce neurons, glia, pigment cells, and connective tissue in vivo and in vitro. To test the developmental potential of apparently undifferentiated crest cells, we have used the monoclonal antibody A2B5, which recognizes a cell surface glycolipid characteristic of neurons, to identify and immunoablate a subpopulation of cultured avian neural crest cells with a neuronal phenotype. Our results indicate that a limited neurogenic precursor subpopulation is present in cultures of avian neural crest cells and that the fate of this subpopulation can be influenced by environmental conditions arising when dispersal of neural crest cells is delayed.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.     

Neural crest cell lineage segregation in the mouse neural tube

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.     

The early steps of neural crest development.

The neural crest is an intriguing cell population that gives rise to many derivatives which are all generated far from their final destinations. From its induction to the delamination of the cells, multiple signalling pathways converge to regulate the expression of effector genes, the products of which endow the cells with invasive and migratory properties reminiscent of those displayed by malignant cells in tumours. As such, the neural crest constitutes an excellent model to study cell migration.     

Restriction of neurogenic ability during neural crest cell differentiation

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells. © 1993 John Wiley & Sons, Inc.     

Cell cycle of neural crest cells in the early migratory stage in vivo

Abstract. The fact that directional migration of neural crest cells (NCC) in vivo occurs in narrow pathways at high cell density, together with our preliminary results showing their high proliferative behaviour, supports the view that a 'population pressure' could be an important factor in the mechanism of early dispersion of NCC.
The purpose of this work was to establish the cranial proliferative pattern of chick embryo NCC during their early migratory stage in vivo . Growth rates and parameters of cell cycle were obtained from cell populations at several cephalic levels by means of autoradiography after labelling with [³H]dT. The labelled cell index of NCC (Forebrain, 0.288; Midbrain, 0.206; and Hindbrain, 0.134) was significantly greater than in other cells populations (e.g. for the neural tube cells: 0.085, 0.030, and 0.031, respectively). The cell generation time was the shortest in NCC (16 h), compared to ectoderm (33 h), mesoderm (58 h) and endoderm (72 h). The duration of the cell cycle phases for NCC were: M, 0.29 h; G₁, 11.23 h; S, 3.40 h; and G₂, 1.02 h.
These quantitative results show that NCC have the greatest proliferative rate in young chick embryos. In relation to cranial regions, the data are consistent with the idea that, in the early migratory phase of this cell population in vivo , migration is in part driven by population pressure.     

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.     

Specification and formation of the neural crest: Perspectives on lineage segregation

The neural crest is a fascinating embryonic population unique to vertebrates that is endowed with remarkable differentiation capacity. Thought to originate from ectodermal tissue, neural crest cells generate neurons and glia of the peripheral nervous system, and melanocytes throughout the body. However, the neural crest also generates many ectomesenchymal derivatives in the cranial region, including cell types considered to be of mesodermal origin such as cartilage, bone, and adipose tissue. These ectomesenchymal derivatives play a critical role in the formation of the vertebrate head, and are thought to be a key attribute at the center of vertebrate evolution and diversity. Further, aberrant neural crest cell development and differentiation is the root cause of many human pathologies, including cancers, rare syndromes, and birth malformations. In this review, we discuss the current findings of neural crest cell ontogeny, and consider tissue, cell, and molecular contributions toward neural crest formation. We further provide current perspectives into the molecular network involved during the segregation of the neural crest lineage.     

The stem cells of the neural crest

In the vertebrate embryo, the neurectodermal neural crest cells (NCC) have remarkably broad potencies, giving rise, after a migratory phase, to neurons and glial cells in the peripheral nervous system, and to skin melanocytes, being all designated here as “neural” derivatives. NC-derived cells also include non-neural, “mesenchymal” cell types like chondrocytes and bone cells, myofibroblasts and adipocytes, which largely contribute to the head structures in amniotes. Similar to the blood cell system, the NC is therefore a valuable model to investigate the mechanisms of cell lineage diversification in vertebrates. Whether NCC are endowed with multiple differentiation potentials or if, conversely, they are a mosaic of different committed cells is an important ongoing issue to understand the ontogeny of NC derivatives in normal development and pathological conditions. Here we focus on recent findings that established the presence in the early migratory NC of the avian embryo, of a multipotent progenitor endowed with both mesenchymal and neural differentiation capacities. This “mesenchymal-neural” clonogenic cell lies upstream of all the other NC progenitors known so far and shows increased frequency when single cell cultures are treated with the Sonic Hedgehog signaling molecule. These findings are discussed in the context of the broad potentials of NC stem cells recently evidenced in certain adult mammalian tissues.     

Growth factor synergism and antagonism in early neural crest development.

This review article focuses on data that reveal the importance of synergistic and antagonistic effects in growth factor action during the early phases of neural crest development. Growth factors act in concert in different cell lineages and in several aspects of neural crest cell development, including survival, proliferation, and differentiation. Stem cell factor (SCF) is a survival factor for the neural crest stem cell. Its action is neutralized by neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) through apoptotic cell death. In contrast, SCF alone does not support the survival of melanogenic cells (pigment cell precursors). They require the additional presence of a neurotrophin (NGF, BDNF, or NT-3). Fibroblast growth factor-2 (FGF-2) is an important promoter of proliferation in neuronal progenitor cells. In neural crest cells, fibroblast growth factor treatment alone does not lead to cell expansion but also requires the presence of a neurotrophin. The proliferative stimulus of the fibroblast growth factor - neurotrophin combination is antagonized by transforming growth factor beta-1 (TGFbeta-1). Moreover, TGFbeta-1 promotes the concomitant expression of neuronal markers from two cell lineages, sympathetic neurons and primary sensory neurons, indicating that it acts on a pluripotent neuronal progenitor cell. Moreover, the combination of FGF-2 and NT3, but not other neurotrophins, promotes expression or activation of one of the earliest markers expressed by presumptive sympathetic neuroblasts, the norepinephrine transporter. Taken together, these data emphasize the importance of the concerted action of growth factors in neural crest development at different levels and in several cell lineages. The underlying mechanisms involve growth-factor-induced dependence of the cells on other factors and susceptibility to growth-factor-mediated apoptosis.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.