Lineage Segregation Research Articles

Identification of a homologue of POU5F1 (OCT3/4) in a marsupial, the brushtail possum.

Several morphological features of early development differ between marsupials and eutherians. These include early overt conceptus polarity, the mode of segregation of pluriblast and trophoblast lineages, and the superficial position of the pluriblast within the unilaminar blastocyst epithelium. Knowledge of the molecular mechanisms of early development is lacking in marsupials. We have cloned from the brushtail possum, Trichosurus vulpecula, the partial cDNA of a marsupial homologue of mammalian POU5F1, which encodes octamer-binding transcription factor-4 (Oct-4). The 579 bp cloned coding sequence of possum POU5F1 (tvPOU5F1) shares 74, 78, and 79% identity with murine Pou5f1, human POU5F1, and bovine bPOU5F1, respectively, at the protein level. The mRNA for tvPOU5F1 was detected by in situ hybridization in oogonia and in oocytes of growing follicles, but not in early meiotic oocytes. In a gastrulation-stage conceptus, high tvPOU5F1 expression was found in the embryonic ectoderm, with low or undetectable expression in other cell types. These results are comparable with those of murine and bovine expression studies, and suggest that the putative role of POU5F1 in totipotent and pluripotent cells is conserved among mammals.

(R)evolutionary considerations in hematopoietic development.

Evolutionary aspects of three characteristics of the mammalian hematopoietic system are considered in the context of both established and recent data. First, the lineage relationships among early members of the hematopoietic hierarchy are reconsidered in a tripartite model proposing lineage segregation based on vascular function, innate immunity, and acquired immunity on an evolutionary time scale. Second, the observation of two stem cell populations that differ in cell cycle status is considered as an evolved mechanism to enhance survival of the species in response to exposure to environmental toxins. Finally, the mobilization of hematopoietic stem cells into the peripheral circulation is proposed to be a mechanism for rapid dissemination of myeloid function during acute bacterial infections. These revolutionary hypotheses challenge some conventional concepts of stem cell biology, and provide an evolutionary context for considering mammalian hematopoiesis.

Identification of early postmitotic cells in distinct embryonic sites and their possible roles in morphogenesis.

Differential proliferation within defined embryonic anlage is likely to play a major role in morphogenesis. We have identified cell populations in the avian embryo that begin exiting the cell cycle as early as the 25-somite stage. These include first the floor plate and then the roof plate of the neural tube, cells that constitute the lamina terminalis and the diencephalic-mesencephalic junction of the developing brain. Outside the nervous system, the central portion of the notochord contains early postmitotic cells. In the heart, such cells will populate the epimyocardium at the level of the truncus arteriosus exclusively and the endocardial cushions that serve as an anchor for the growing intracardial septa. Surprisingly, the endoderm at the level of the prospective midgut is composed of post-mitotic progenitors. These cells are later found both in the caudal portion of the duodenum and in derivatives adjacent to the umbilical region of the primitive midgut. The possible implications of this early, localized withdrawal from the cell cycle to morphogenetic events and lineage segregation are discussed.

A genetic screen for novel components of the notch signaling pathway during Drosophila bristle development.

The Notch receptor is the central element in a cell signaling mechanism controlling a broad spectrum of cell fate choices. Genetic modifier screens in Drosophila and subsequent molecular studies have identified several Notch pathway components, but the biochemical nature of signaling is still elusive. Here, we report the results of a genetic modifier screen of the bristle phenotype of a gain-of-function Notch allele, Abruptex16. Abruptex mutations interfere with lateral inhibition/specification events that control the segregation of epidermal and sensory organ precursor lineages, thus inhibiting bristle formation. Mutations that reduce Notch signaling suppress this phenotype. This screen of approximately 50,000 flies led to the identification of a small number of dominant suppressors in seven complementation groups. These include known components in the pathway, Notch, mastermind, Delta, and Hairless, as well as two novel mutations. The first, A122, appears to interact with Notch only during bristle development. The other, M285, displays extensive genetic interactions with the Notch pathway elements and appears, in general, capable of suppressing Notch gain-of-function phenotypes while enhancing Notch loss-of-function phenotypes, suggesting that it plays an important role in Notch signaling.

Induction and patterning of the neural crest, a stem cell-like precursor population.

The neural crest is a multipotent precursor population which ultimately generates much of the peripheral nervous system, epidermal pigment cells, and a variety of mesectodermal derivatives. Individual multipotent neural crest cells are capable of some self-renewing divisions, and based upon this criteria can be considered stem cells. Considerable progress has been made in recent years toward understanding how this important population of progenitor cells is initially established in the early embryo, and how cell-intrinsic and non-cell-intrinsic factors mediate their subsequent lineage segregation and differentiation.

Population structure of nuclear and mitochondrial DNA variation among humpback whales in the North Pacific.

The population structure of variation in a nuclear actin intron and the control region of mitochondrial DNA is described for humpback whales from eight regions in the North Pacific Ocean: central California, Baja Peninsula, nearshore Mexico (Bahia Banderas), offshore Mexico (Socorro Island), southeastern Alaska, central Alaska (Prince Williams Sound), Hawaii and Japan (Ogasawara Islands). Primary mtDNA haplotypes and intron alleles were identified using selected restriction fragment length polymorphisms of target sequences amplified by the polymerase chain reaction (PCR-RFLP). There was little evidence of heterogeneity in the frequencies of mtDNA haplotypes or actin intron alleles due to the year or sex composition of the sample. However, frequencies of four mtDNA haplotypes showed marked regional differences in their distributions (phi ST = 0.277; P < 0.001; n = 205 individuals) while the two alleles showed significant, but less marked, regional differences (phi ST = 0.033; P < 0.013; n = 400 chromosomes). An hierarchical analysis of variance in frequencies of haplotypes and alleles supported the grouping of six regions into a central and eastern stock with further partitioning of variance among regions within stocks for haplotypes but not for alleles. Based on available genetic and demographic evidence, the southeastern Alaska and central California feeding grounds were selected for additional analyses of nuclear differentiation using allelic variation at four microsatellite loci. All four loci showed significant differences in allele frequencies (overall FST = 0.043; P < 0.001; average n = 139 chromosomes per locus), indicating at least partial reproductive isolation between the two regions as well as the segregation of mtDNA lineages. Although the two feeding grounds were not panmictic for nuclear or mitochondrial loci, estimates of long-term migration rates suggested that male-mediated gene flow was several-fold greater than female gene flow. These results include and extend the range and sample size of previously published work, providing additional evidence for the significance of genetic management units within oceanic populations of humpback whales.

Segregation of myogenic lineages in Drosophila requires numb.

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that found larval muscles or form precursors of adult muscles. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. These differences depend on an asymmetric segregation of Numb between sibling cells. In numb mutants, muscle fates associated with repression are duplicated and alternative muscles are lost. If numb is overexpressed the reverse transformation occurs. Numb acts to block Notch-mediated repression of genes expressed in muscle progenitor cells. Thus asymmetric cell divisions are essential determinants of muscle fates during myogenesis in Drosophila

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation.

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.

The neural crest population responding to endothelin-3 in vitro includes multipotent cells.

The peptide endothelin 3 (EDN3) is essential for normal neural crest development in vivo, and is a potent mitogen for quail truncal crest cells in vitro. It is not known which subpopulations of crest cells are targets for this response, although it has been suggested that EDN3 is selective for melanoblasts. In the absence of cell markers for different precursor types in the quail crest, we have characterised EDN3-responsive cell types using in vitro colony assay and clonal analysis. Colonies were analysed for the presence of Schwann cells, melanocytes, adrenergic cells or sensory-like cells. We provide for the first time a description of the temporal pattern of lineage segregation in neural crest cultures. In the absence of exogenous EDN3, crest cells proliferate and then differentiate. Colony assay indicates that in these differentiated cultures few undifferentiated precursors remain and there is a low replating efficiency. By contrast, in the presence of 100 ng/ml EDN3 differentiation is inhibited and most of the cells maintain the ability to give rise to mixed colonies and clones containing neural crest derivatives. A high replating efficiency is maintained. In secondary culture there was a progressive decline in the number of cell types per colony in control medium. This loss of developmental potential was not seen when exogenous EDN3 was present. Cell type analysis suggests two novel cellular targets for EDN3 under these conditions. Contrary to expectations, one is a multipotent precursor whose descendants include melanocytes, adrenergic cells and sensory-like cells; the other can give rise to melanocytes and Schwann cells. Our data do not support previous claims that the action of EDN3 in neural crest culture is selective for cells in the melanocyte lineage.

1 The Avian Embryo as a Model in Developmental Studies: Chimeras and in Vitro Clonal Analysis

The avian embryo is a model in which techniques of experimental embryology and cellular and molecular biology can converge to address fundamental questions of development biology. The first part of the chapter describes two examples of transplantation and cell labeling experiments performed in ovo. Thanks to the distinctive histologic and immunocytochemical characteristics of quail and chick cells, the migration and development of definite cells are followed in suitably constructed chimeric quail-chick embryos. Isotopic transplantations of neural tube portions between quail and chick, combined with in situ hybridization with a nucleic probe specific for a quail oligodendrocyte marker, allowed study of the origin and migration of oligodendroblasts in the spinal cord. Heterotopic transplantations of rhombomeres were performed to establish the degree of plasticity of these segments of the hindbrain regarding Hox gene expression, which was revealed by labeling with chick-specific nucleic probes. The second part describes in vitro cell cloning experiments devised to investigate cell lineage segregation and diversification during development of the NC. An original cloning procedure and optimal culture conditions permitted analysis of the developmental potentials of individual NC cells taken at definite migration stages. The results revealed a striking heterogeneity of the crest cell population, which appeared to be composed of precursors at different states of determination. Clonal cultures also provide a means to identify subsets of cells that are the target of environmental factors and to understand how extrinsic signals influence the development of responsive cells.

First and second cycle nuclear transfer in cattle: Comparison of efficiency in embryo development and ongoing pregnancies

The first lineage segregation in the pre-implantation mouse embryo gives rise to cells of the inner cell mass and the trophectoderm. Segregation into these two lineages during the 8-cell to 32-cell stages is accompanied by a significant amount of cell displacement, and as such it has been difficult to accurately track cellular behavior using conventional imaging techniques. Consequently, how cellular behaviors correlate with cell fate choices is still not fully understood. To achieve the high spatial and temporal resolution necessary for tracking individual cell lineages, we utilized two-photon light-scanning microscopy (TPLSM) to visualize and follow every cell in the embryo using fluorescent markers. We found that cells undergoing asymmetric cell fate divisions originate from a unique population of cells that have been previously classified as either outer or inner cells. This imaging technique coupled with a tracking algorithm we developed allows us to show that these cells, which we refer to as intermediate cells, share features of inner cells but exhibit different dynamic behaviors and a tendency to expose their cell surface in the mouse embryo between the fourth and fifth cleavages. We provide an accurate description of the correlation between cell division order and cell fate, and demonstrate that cell cleavage angle is a more accurate indicator of cellular polarity than cell fate. Our studies demonstrate the utility of two-photon imaging in answering questions in the pre-implantation field that have previously been difficult or impossible to address. Our studies provide a framework for the future use of specific markers to track cell fate molecularly and with high accuracy.

Genomic regions regulating early embryonic expression of the Drosophila neurogenic gene Delta

To identify genomic regions required for transcriptional regulation of Delta during early embryogenesis, constructs carrying promoter and gene fusions to the lacZ gene were used for germ line transformation. Most cis-regulatory sequences are dispersed throughout 6.6. kb of genomic DNA, 5′ of the transcription start site; the first intron contains an enhancer element that increases the amount of RNA produced in several organs. A region was defined which drives Delta-lacZ RNA expression in clusters of neuroectodermal cells preceding and during SI neuroblast segregation. This pattern is regulated by genes of the AS-C ( achaete-scute complex). To identify regulatory regions necessary for normal function of Delta during neural-epidermal lineage segregation, five minigenes consisting of fragments of the 5′ genomic DNA fused to a cDNA encoding the entire protein sequence were tested for their ability to rescue the neural hyperplasia caused by a deletion of the Delta locus. Regulatory sequences required for this function are differentially distributed throughout 9 kb of genomic DNA upstream of the transcription start site. The possible significance of these findings with respect to the function of Delta during lineage segregation is discussed.

Stem cells and transcription factors in the development of the mammalian neural crest.

The neural crest is a migratory population of multipotent embryonic cells that generates the neurons and glia of the peripheral nervous system, as well as a variety of non-neural mesectodermal and endocrine cell types. The study of neural crest cell and molecular biology provides a system to investigate how such multipotent cells choose their fates, and whether the repertoire of fates becomes progressively restricted with time. The study of mammalian neural crest development has lagged behind studies of avian crest development due to the relative inaccessibility of mammalian embryos. The development of reverse genetic methods in mice, however, has made the analysis of mammalian neural crest development both more attractive and more tractable. Rodent neural crest cells have been isolated and grown in clonogenic cultures, where they behave as multipotent stem cells. This system provides an assay for factors that influence the differentiation of these multipotent cells. Transcription factors provide valuable early markers for neural crest cells as well as molecular handles on the lineage segregation process. One such factor is Mash1, a homolog of the Drosophila proneural genes, achaete-scute. Mash1 marks autonomic progenitor cells and is essential for their development in vivo, as shown by gene knockout experiments.

From the Crest to the Periphery: Control of Pigment Cell Migration and Lineage Segregation

Pigment cells are one of many cell types derived from the neural crest. This review focuses on the mechanisms that control the timing and pathways of migration of pigment cells into the epidermis and determinants that control the differentiation of pigment cells. Several factors may control the timing and pattern of pigment cell migration in the dorsolateral space including the loss of inhibitory molecules in the pathway, the appearance of chemotactic molecules emanating from the dispersing dermatome, and the differentiation of pigment cells, which may be the only neural crest derivative capable of utilizing the substratum found in the dorsolateral path. Control of pigment cell differentiation remains controversial. A working model presented in this review suggests that multipotent neural crest cells that disperse ventrally upon separation from the neural tube preserve neurogenic ability and lose melanogenic ability, whereas those cells that are arrested at the entrance to the dorsolateral path lose neurogenic ability so that the population becomes primarily melanogenic. During the time that the latter population is arrested in migration it is speculated that the neural crest cells are exposed to an environment comprised of specific extracellular matrix molecules and/or growth factors that enhance pigment cell differentiation.

In Vitro Clonal Analysis of Mouse Neural Crest Development

Analysis of lineage segregation during mammalian neural crest development has not been sufficiently performed due to technical difficulties. In the present study, therefore, we established a clonal culture system of mouse neural crest cells in order to analyze developmental potentials of individual neural crest cells and their patterns of lineage segregation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin (CT) were applied to culture medium to trigger melanogenic differentiation of mouse neural crest cells. Three morphologically distinct types of clones were observed. (1) "Pigmented clones" consisted of melanocytes only, suggesting that the clone-forming cells were committed to the melanogenic lineage. These clones were observed only in the presence of TPA and CT. The proportion of this type of clone (8%) was much lower than that of the equivalent type of clone in quail trunk neural crest (40-60%; Sieber-Blum and Cohen, 1980, Dev. Biol. 80, 96-106). It therefore appears that the segregation pattern to the melanogenic lineage during mouse neural crest development in vitro differs quantitatively from that in the quail. (2) "Mixed clones" consisted of pigmented and unpigmented cells. Like pigmented clones, they were observed only in the presence of TPA and CT. The clones contained up to four types of cells: melanocytes, S100-positive cells (Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive autonomic neuron-like cells, and substance P (SP)-immunoreactive sensory neuron-like cells. Thus, at least some mixed clone-forming cells are pluripotent. (3) Two classes of "unpigmented clones" were observed that consisted of unpigmented cells only. These clones developed in the presence and absence of TPA and CT. Unpigmented clones in one class contained up to three types of cells as well as other, as yet unidentified cells: S100-, 5-HT-, and SP-positive cells. This observation suggests that at least some of these clones originate from cells with a partially restricted developmental potential. Clones in another class consisted of S100- or SP-positive cells only. These clones might be derived from cells restricted to the SP-positive neuronal cell or melanocyte/Schwann cell lineage. The present data indicate that at initiation of migration, the mouse neural crest of the trunk region is a heterogeneous population of cells containing pluripotent cells, cells with a restricted developmental potential, and cells apparently committed to the melanogenic cell lineage.

Pluripotent and Developmentally Restricted Neural-Crest-Derived Cells in Posterior Visceral Arches

The early migratory cells of the posterior rhombencephalic neural crest consist of a heterogeneous population of pluripotent and developmentally restricted neural crest cells (Ito and Sieber-Blum, Dev. Biol. 148, 95-106, 1991). To determine if changes in developmental capacities occur during migration, the developmental potentials of posterior visceral arch mesenchymal cells were investigated by in vitro clonal analysis. Most of these cells consisted of the post-migratory cells of the posterior rhombencephalic neural crest. Four morphologically distinct types of clones were observed, and the cells within these clones expressed characteristic phenotypes as shown by their binding of antibodies against cell type-specific markers: (1) "DP" clones consisted of densely packed polygonal cells, with flattened large cells located predominantly at the periphery of these clones. Immunocytochemical analyses showed that DP clones contained smooth muscle cells, connective tissue cells, chondrocytes, and serotonin (5-HT)-positive neurons, and over 90% of the cells per clone were HNK-1 positive. This suggests that DP clone-forming cells are pluripotent neural-crest-derived cells with the capacity to develop into ectomesenchymal derivatives and serotonergic neurons. (2) "DS" clones consisted of densely packed spindle-shaped cells. These clones included smooth muscle cells, connective tissue cells, and chondrocytes. By contrast, neuronal phenotypes were not present. An average of 0.4% of the cells per clone were HNK-1 positive. DS clones appear to be formed by neural-crest-derived cells that are partially restricted, expressing ectomesenchymal phenotypes only. (3) "DF" clones consisted of densely packed small cells and flattened large cells. Although no HNK-1-positive cells were found in DF clones, these clones contained connective tissue cells and/or smooth muscle cells. DF clones appear to be derived from bipotent cells with the ability to differentiate into connective tissue cells and smooth muscle cells, or cells committed to the connective tissue cell lineage. (4) "LF" clones consisted of loosely arranged, flattened large cells. These clones did not contain HNK-1-positive cells. The clones consisted entirely of smooth muscle cells. Therefore, LF clones are most likely formed by precursors that are committed to the smooth muscle cell lineage. These results indicate the presence of pluripotent neural-crest-derived cells, cells with a restricted developmental potential, and apparently committed cells in the posterior visceral arch. Pluripotent cells can generate up to four neuronal and nonneuronal phenotypes. Other cells are restricted to ectomesenchymal cell types, and the proportion of these cells in the posterior visceral arch changes with progressing embryonic development. The present data in combination with our previous study (Ito and Sieber-Blum, Dev. Biol. 148, 95-106, 1991) suggest a characteristic pattern of lineage segregation during posterior rhombencephalic neural crest development, indicating that specification of some cell types can occur at the final sites of the neural crest cell localization.

Expression of spatially regulated genes in the sea urchin embryo

Spatially controlled genes expressed in the early sea urchin embryo have been characterized, and the patterns of expression in terms of the mechanisms by which this embryo accomplishes its initial set of founder cell specifications are the subject of current discussion. Sea urchin transcription factors that have been cloned are classified with respect to their target sites and the genes they regulate. Among the best known of the sea urchin cis-regulatory systems is that controlling expression of the Cyllla gene, which encodes an aboral ectoderm-specific cytoskeletal actin. The Cyllla regulatory domain includes approximately 20 sites of DNA-protein interaction, serviced by about ten different factors. Certain of these factors are known to negatively control spatial expression, while others positively regulate temporal activation and the level of Cyllla gene expression. Differential, lineage-specific gene expression is instituted in the sea urchin embryo by mid-late cleavage, prior to any cell migration or overt differentiation, and shortly following lineage segregation.

Expression of spatially regulated genes in the sea urchin embryo

Spatially controlled genes expressed in the early sea urchin embryo have been characterized, and the patterns of expression in terms of the mechanisms by which this embryo accomplishes its initial set of founder cell specifications are the subject of current discussion. Sea urchin transcription factors that have been cloned are classified with respect to their target sites and the genes they regulate. Among the best known of the sea urchin cis-regulatory systems is that controlling expression of the Cyllla gene, which encodes an aboral ectoderm-specific cytoskeletal actin. The Cyllla regulatory domain includes approximately 20 sites of DNA-protein interaction, serviced by about ten different factors. Certain of these factors are known to negatively control spatial expression, while others positively regulate temporal activation and the level of Cyllla gene expression. Differential, lineage-specific gene expression is instituted in the sea urchin embryo by mid-late cleavage, prior to any cell migration or overt differentiation, and shortly following lineage segregation.

In Search of Molecular Origins of Cellular Differentiation in Volvox and Its Relatives

Publisher Summary This chapter presents a summary of the morphological and biochemical evidence that supports the volvocine-lineage hypothesis. It describes attempts to evaluate the validity of this hypothesis objectively with molecular phylogenetic methods. The chapter also reviews advances that have occurred in three areas of Volvox research:(1) the genetic and molecular control of germ or soma differentiation, (2) the mechanism of action of the sexual pheromone, and (3) the comparative anatomy and evolution of selected structural genes of Volvox and Chlamydomonas. The volvocine algae (Chlamydomonas and the members of the family Volvocaceae) all have a fundamentally similar and distinctive pattern of subcellular organization. However, the volvocine algae can be conceptually lined up in a series (Chlamydomonas, Gonium, Pandorina, Eudorina, Pleodorina, and Volvox), in which there is a progressive increase in each of the parameters—cell number, organismic size, tendency for some cells to differentiate terminally as somatic cells, and ratio of somatic-cell number to reproductive-cell number. The volvocine lineage hypothesis postulates that this conceptual series may actually reflect the evolutionary history of the group. The aspect of Volvox carteri development that is most interesting from an evolutionary genetic standpoint is the aspect that most clearly differentiates it from other genera of volvocine algae, and from most other species of Volvox, i.e., the early and complete segregation of separate germ and somatic-cell lineages.

V- myc immortalization of early rat neural crest cells yields a clonal cell line which generates both glial and adrenergic progenitor cells

We describe the isolation and characterization of an immortal cell line derived by infection of rat neural crest cells with a v- myc-containing replication-defective retrovirus. This clonal cell line, called NCM-1, contains a majority cell population with antigenic and morphologic properties that suggest it may represent a peripheral glial progenitor. In conditioned or in serum-free medium, these NGF receptor-positive cells differentiate to an elongated, bipolar morphology resembling that of primary Schwann cells. This morphologic differentiation is prevented by TGF- β 1, which also acts as a mitogen for the cells. The NCM-1 line is also able to generate clonal derivatives which have extinguished expression of most or all glial markers. Once generated, such cells are stable and do not revert to the glial phenotype. At least some of these cells have acquired sympathoadrenal progenitor-like properties, as shown by their capacity to coexpress tyrosine hydroxylase (TH) and neurofilament (NF) in response to basic FGF and dexamethasone. These data imply that the NCM-1 line contains self-renewing cells with the potential to generate precursors in at least two of the sublineages that normally develop from the neural crest. This in turn suggests that the process of immortalization may preserve at least some of the developmental properties characteristic of multipotential neural crest cells. NCM-1 cells may prove useful for the study of neural crest cell lineage segregation, Schwann cell differentiation, and the mechanisms controlling the initial induction of TH and NF gene expression.