Exogenous basement-membrane-like matrix stimulates adrenergic development in avian neural crest cultures

The development of quail trunk neural crest cultures was dramatically altered when the cultures were overlaid with a gel of reconstituted basement membrane (RBM) components derived from the Engelbreth-Holm-Swarm sarcoma. In the presence of the RBM gel overlay, the number of catecholamine-positive (CA+) cells that developed was increased 50-fold, while the final number of melanocytes and total cells was only half that seen in the control cultures. The presence of the RBM gel overlay did not alter the time of onset of differentiation of the CA+ cells or melanocytes. The stimulation of CA+ cell number was not observed with type IV collagen substrates, laminin substrates or type I collagen gel overlays with or without added laminin. The stimulation of CA+ cell development was dependent on initial plating density. The number of CA+ cells that developed in the presence of the RBM gel was proportional to the initial plating density at 80-320 cells mm-2, whereas no CA+ cells were observed below 20 cells mm-2 and only a few CA+ cells were detected at 40 cells mm-2. There was, however, extensive cell division and differentiation of melanocytes and unpigmented cells at the lower initial plating densities. When the RBM gel was used as a substrate, rather than as an overlay, a striking rearrangement of cells into interconnected strands was observed. After several days in culture, melanocytes, CA+ cells and unpigmented cells were present in these strands. These results indicate that molecules associated with a reconstituted basement-membrane-like matrix are a potent stimulatory influence on adrenergic development and also act to inhibit the production of other cell types in neural crest cultures.     

An Analysis of the Effects of Retinoic Acid and Other Retinoids on the Development of Adrenergic Cells from the Avian Neural Crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-transRA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-transRA and 13-cisRA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-transRA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-α, produced an increase in adrenergic cells similar to that seen with all-transRA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-transRA is mediated by RAR-α and possibly RAR-β. To further define the actions of all-transRA on the neural crest we incubated cultures with 5-bromo-2′-deoxyuridine (BrdU) to determine whether all-transRA could affect the rate of proliferation. The results show that while all-transRA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-transRA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.     

Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells

To determine if neural crest cells are pluripotent and establish whether differentiation occurs in the absence of noncrest cells, a cell culture method was devised in which differentiation could be examined in clones derived from single, isolated neural crest cells. Single neural crest cells, which were isolated before the onset of in vivo migration, gave rise to three types of clones: pigmented, unpigmented, and mixed. Pigmented clones consisted of melanocytes only, whereas some unpigmented cells in mixed and unpigmented clones contained catecholamines, identifying them as adrenergic cells. Extracellular matrix derived from quail somite or chick skin fibroblast cultures stimulated adrenergic differentiation and axon formation. These results demonstrate for the first time the existence of pluripotent quail neural crest cells that give rise to at least two progeny, melanocytes and neuronal cells. They also suggest that continuous direct interactions with noncrest cells are not required for the differentiation of these two cell types. However, components of the extracellular matrix derived from noncrest cells may play an important role in expression of the adrenergic phenotype.     

Neural tube-derived factors influence differentiation of neural crest cells in vitro: effects on activity of neurotransmitter biosynthetic enzymes

Previously, we have demonstrated that a factor present in chick embryo extract or medium conditioned by neural tube cells supports adrenergic differentiation of some neural crest cells in vitro. These studies have been extended here to examine the effects of this factor(s) on the development of enzymes involved in neurotransmitter biosynthesis. The time course of expression of choline acetyltransferase (ChAT), a marker for cholinergic cells, and dopamine-beta-hydroxylase (DBH), a marker for adrenergic cells, was examined in neural crest cell cultures grown under three conditions: in medium containing 10% embryo extract, in medium containing 2% embryo extract, and in medium containing 2% embryo extract that was conditioned by neural tube cells (NTCM). Significant levels of DBH activity were measured in neural crest cell cultures grown in 10% embryo extract containing medium or in NTCM, while only low levels were present in cultures grown in medium containing 2% embryo extract. In contrast, ChAT activity was inhibited by NTCM in comparison to levels in both 10 and 2% embryo extract containing medium. As a preliminary characterization of the factor(s) present in chick embryo extract, we have fractionated embryo extract and find that a pool of 10 kDa or less can support adrenergic differentiation of some neural crest cells. These results suggest that low molecular weight factors present in embryo extract and NTCM support adrenergic expression of neural crest cells, whereas NTCM suppresses cholinergic expression.     

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations.

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-(1+)-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1+ cells compared to the remaining HNK-1+ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1+ subpopulations were grown together with HNK-1- cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1+ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1- cells. In the second part of this study, we investigate the specificity of differentiation of HNK-(1+)- and HNK-(1-)-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-(1+)-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-(1-)-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-(1+)- and the HNK-(1-)-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.     

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.     

Inhibition of the adrenergic phenotype in cultured neural crest cells by norepinephrine uptake inhibitors

Tricyclic antidepressants in combination with in vitro clonal analysis of quail neural crest cells were used to examine the role the norepinephrine uptake mechanism might play in the development of adrenergic neural crest derivatives. Norepinephrine (NE) uptake inhibitors blocked expression of the adrenergic phenotype by neural crest cells. The degree of inhibition of phenotypic expression correlated with the potency and specificity of the uptake inhibitors. The drugs acted during the early phase of in vitro development, i.e., several days before overt expression of the adrenergic phenotype in clonal culture. They were nontoxic, and a chronic exposure of the cells to NE uptake inhibitors was necessary to cause an effect. These observations suggest that norepinephrine and possibly related neurotransmitters play a direct or indirect role in the expression of the adrenergic phenotype by neural crest cells and that tricyclic antidepressants may affect neurogenesis during sensitive stages of embryonic development. The data may reflect in vivo mechanisms, since there are neurotransmitters present in the migratory pathway of presumptive sympathetic neurons and the norepinephrine uptake system is expressed in the embryo by these cells before they synthesize and accumulate catecholamines.     

Partial restriction in the developmental potential of late emigrating avian neural crest cells.

Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. "Early" and "middle" aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, "late" neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10-14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14-17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI-labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population.     

Cultured quail neural crest cells attain competence for terminal differentiation into melanocytes before competence to terminal differentiation into adrenergic neurons

At the onset of migration the quail neural crest contains pluripotent progenitor cells that give rise to both melanocytes and adrenergic neurons as well as progenitor cells that are already committed to the melanogenic or the neuronal pathway. In this paper we show that melanogenic progenitors attain the competence for terminal differentiation prior to adrenergic progenitors. The adrenergic phenotype was only expressed when the crest cells were allowed to proliferate in vitro for at least 3 days. Differentiation into melanocytes, however, occurred even when proliferation was blocked with cytosine arabinoside immediately after explantation of the neural tube.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Extracellular matrix from normal but not Steel mutant mice enhances melanogenesis in cultured mouse neural crest cells

The Steel mutation is a non-cell-autonomous defect in mice that affects the development of several stem cell populations, including germ cells, hematopoietic cells, and neural crest-derived pigment cells. To characterize the environmental lesion caused by the Steel mutation, we have compared the ability of normal and mutant extracellular matrix material to support the differentiation of normal mouse neural crest cells in vitro. Extracellular matrix deposited by cultured skin cells isolated from normal fetuses enhanced melanogenesis by crest cells over that observed on plastic substrata. In contrast, matrix material produced by Steel-Dickie (Sld) fetal skin cells failed to enhance melanogenesis. Adrenergic differentiation by neural crest-derived cells was promoted equally by both normal and mutant extracellular matrix compared to control substrata. We conclude that the environmental defect in mutant embryos selectively affects a melanogenic subpopulation of neural crest cells and resides, at least in part, in the extracellular matrix.     

Cyclic AMP signaling functions as a bimodal switch in sympathoadrenal cell development in cultured primary neural crest cells

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development of the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures. To investigate whether interactive mechanisms between signaling pathways influence crest cell differentiation, we characterized the combinatorial effects of BMP-2 and cAMP-elevating agents on the development of quail trunk neural crest cells in primary culture. We report that the cAMP signaling pathway modulates both positive and negative signals influencing the development of SA cells. Specifically, we show that moderate activation of cAMP signaling promotes, in synergy with BMP-2, SA cell development and the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and the expression of the SA lineage-determining ASH-1 and Phox2 genes. We conclude that cAMP signaling acts as a bimodal regulator of SA cell development in neural crest cultures.     

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-1⁺-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1⁺ cells compared to the remaining HNK-1⁺ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1⁺ subpopulations were grown together with HNK-1^? cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1⁺ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1^? cells. In the second part of this study, we investigate the specificity of differentiation of HNK-1⁺- and HNK-1^?-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-1⁺-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-1^?-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-1⁺- and the HNK-1^?-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.     

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

Neural crest cells: From developmental biology to clinical interventions

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. © 2014 Wiley Periodicals, Inc.     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

A role for foxd3 and sox10 in the differentiation of gonadotropin-releasing hormone (GnRH) cells in the zebrafish Danio rerio

Gonadotropin-releasing hormone (GnRH) is found in a wide range of vertebrate tissues, including the nervous system. In general, GnRH has two functions: endocrine, acting as a releasing hormone; and neuromodulatory, affecting neural activity in the peripheral and central nervous system. The best understood population of GnRH cells is that of the hypothalamus, which is essential for reproduction. Less well understood are the populations of GnRH cells found in the terminal nerve and midbrain, which appear to be neuromodulatory in function. The GnRH-containing cells of the midbrain are proposed to arise from the mesencephalic region of the neural tube. Previously, we showed that neuromodulatory GnRH cells of the terminal nerve arise from cranial neural crest. To test the hypothesis that neuromodulatory GnRH cells of the midbrain also arise from neural crest, we used gene knockdown experiments in zebrafish to disrupt neural crest development. We demonstrate that decrement of the function of foxd3 and/or sox10, two genes important for the development and specification of neural crest, resulted in a reduction and/or loss of GnRH cells of the midbrain, as well as a reduction in the number of terminal nerve GnRH cells. Therefore, our data support a neural crest origin for midbrain GnRH cells. Additionally, we demonstrate that knockdown of kallmann gene function resulted in the loss of endocrine GnRH cells of the hypothalamus, but not of neuromodulatory GnRH cells of the midbrain and terminal nerve, thus providing additional evidence for separate pathways controlling the development of neuromodulatory and endocrine GnRH cells.     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.