Expression and regulation of type I BMP receptors during early avian sympathetic ganglion development.

We have investigated the expression and regulation of the mRNAs for the type I BMP receptors, BMPR-IA and BMPR-IB, in quail embryos in vivo and in neural crest cultures in vitro. BMPR-IB mRNA was expressed in the primordial sympathetic ganglia at stage 17, soon after the first expression of Cash-1 mRNA, the avian homolog of the Drosophila transcription factor achaete-scute. BMP-4 mRNA was detected in the dorsal aorta at stage 17, coincident with BMPR-IB mRNA expression in the sympathetic ganglia. BMPR-IA mRNA was first expressed in the sympathetic ganglia at stage 18. Moreover, BMP-4 ligand mRNA was detected in the sympathetic ganglia starting at stage 18. BMPR-IA and BMPR-IB were differentially regulated in cultured neural crest cells. BMPR-IB was expressed in primary outgrowths of neural crest cells but was downregulated after primary outgrowths were harvested and replated in secondary cultures. In secondary cultures of neural crest cells, exogenous BMP-2 and BMP-4 increased the expression of BMPR-IA but decreased the expression of BMPR-IB. The expression of both type I BMP receptors was inhibited by exogenous TGF-beta1. Our results suggest distinct roles for BMPR-IA and BMPR-IB in the development of the sympathoadrenal phenotype from cells of the neural crest.

Bone morphogenetic proteins-2 and -4 attenuate apoptosis in a cerebellar primitive neuroectodermal tumor cell line.

Similarities between primitive neuroectodermal tumors and central nervous system (CNS) progenitor cells have evoked interest in the response of these tumors to endogenous growth factors. The bone morphogenetic proteins (BMPs) have recently been found to regulate survival and differentiation of CNS progenitor cell populations. In this study, we investigated the effects of BMP-2, BMP-4, and BMP-6 on the undifferentiated cerebellar primitive neuroectodermal tumor or medulloblastoma cell line DAOY. Analysis by reverse transcriptase-polymerase chain reaction showed that mRNAs for type IA and type II BMP receptors were present in control cultures. In cultures treated with BMP-2, mRNAs for BMP receptor type IB and the activin R-I receptor became evident. Cultures were analyzed for total cell counts, proliferating cell nuclear antigen (PCNA), and apoptotic DNA fragmentation. There was a significant increase in total cell number in the BMP-2 and BMP-4 treatment groups, without any change in PCNA reactivity, and a dramatic decrease in the proportion of apoptotic nuclei at concentrations of BMP-2 and BMP-4 above 5 ng/ml (P<0.001). These effects were not observed with BMP-6, TGF-beta1 or GDNF. These results suggest that the increase in total cell number is due to the attenuation of apoptosis by BMP-2 and BMP-4. The anti-apoptotic effect of BMP-2 and BMP-4 on this neuroectodermal cell line has potential clinical implications for neuroectodermal tumors.

Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures.

Previous work has demonstrated that the bone morphogenetic proteins (BMP)-2, BMP-4, and BMP-7 can promote the development of tyrosine hydroxylase (TH)-positive and catecholamine-positive cells in quail trunk neural crest cultures. In the present work, we showed that mRNA for the type I bone morphogenetic protein receptor IA (BMPR-IA) was present in neural crest cells grown in the absence or presence of BMP-4. We have used a replication-competent avian retrovirus to express a constitutively active form of BMPR-IA in neural crest cells in culture. Cultures grown in the absence of BMP-4 and infected with retrovirus containing a construct encoding this activated BMPR-IA developed five times more TH-immunoreactive and catecholamine-positive cells than uninfected control cultures or cultures infected with virus bearing the wild-type BMPR-IA cDNA. The number of TH-positive cells which developed was dependent on the concentration of virus bearing the activated receptor cDNA used in the experiments. Most TH-positive cells which developed also contained viral p19 protein. Total cell number was not affected by infection with the virus containing the activated receptor construct. The effect of the activated receptor was phenotype-specific since infection with the virus bearing the activated receptor cDNA did not alter the number or morphology of microtubule-associated protein (MAP)2-immunoreactive cells, which are distinct from the TH-positive cell population. These findings are consistent with the observation that MAP2-positive cells are not affected by the presence of BMP-4. Taken together, these results suggest that activity of BMPR-IA is an important element in promoting the development of the adrenergic phenotype in neural crest cultures.

Expression of a quail bHLH transcription factor is associated with adrenergic development in trunk neural crest cultures.

1. Expression of chick type 1 basic helix-loop-helix transcription factor GbHLH1.4 persists in several embryonic regions, including some where neural crest cells differentiate (Helms, J. A., et al., Mech. Dev. 48:93-108, 1994.) We have cloned portions of the quail homologue (designated QbHLH) in order to investigate its expression and possible function in quail neural crest cultures. Three sets of polymerase chain reaction primers were used to amplify cDNA sequences encompassing much of the coding region outside the bHLH domain. Two of the primer sets amplified a single band from all quail and chick tissues tested. The third set of primers produced two bands, differing by a 72-base pair insertion, both of which which were present in all tissues assayed. 2. The quail sequences showed greater than 97% nucleotide identity with GbHLH1.4. In situ hybridization of cultured quail neural crest cells showed expression in some, but not all, cells throughout the first 2 weeks in culture. 3. Tyrosine hydroxylase immunoreactivity correlated particularly well with QbHLH expression, although substantial subpopulations of cells with other phenotypes also express QbHLH. 4. In some cells, only limited regions of the cytoplasm showed hybridization with QbHLH probes, indicating possible mRNA localization. 5. The expression of QbHLH in neural crest cultures is consistent with its role as a relatively widely expressed helix-loop-helix dimerization partner and suggests that it may function by interacting with cell type-specific partners to regulate expression of genes involved in the development and maintenance of several phenotypes.

Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation.

Melanocytes in the skin are derived from the embryonic neural crest. Recently, mutations in endothelin 3 and the endothelin receptor B genes have been shown to result in gross pigment defects, indicating that this signalling pathway is required for melanocyte development. We have examined the effects of endothelins on melanocyte progenitors in cultures of mouse neural crest. Firstly, they stimulate an increase in progenitor number and act synergistically with another factor, Steel factor, in the survival and proliferation of the progenitors. These findings are consistent with findings from mice with natural mutations in the endothelin receptor B gene, which show an early loss of melanocyte progenitors. Secondly, endothelins induce differentiation of the progenitors into fully mature pigmented melanocytes. This finding is consistent with the expression of endothelins in the skin of mice at the initiation of pigmentation. The melanocytes generated in endothelin-treated cultures also become responsive to alpha melanocyte-stimulating hormone, which then acts to regulate the activity of the pigmentation pathway. These findings indicate two key roles for endothelin in melanocyte development: regulation of expansion of the progenitor pool and differentiation of progenitors into mature melanocytes.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures.

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) beta 1, TGF-beta 2, and TGF-beta 3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Thyroid hormone decreases the number of adrenergic cells that develop in neural crest cultures and can inhibit the stimulatory action of retinoic acid.

Environmental cues are known to be important in the migration, survival, and differentiation of neural crest cells and their derivatives. Retinoic acid (RA) can increase the number of adrenergic cells that develop in neural crest cultures in a dose dependent manner. These results with RA prompted us to investigate the effects of other retinoids and other related compounds on neural crest cultures. We have investigated the role of thyroid hormone (T3) in the development of adrenergic cells in quail neural crest cultures. T3 produced a significant decrease in the number of catecholamine-positive cells that developed in neural crest cultures after 7 days in vitro, as compared to untreated controls. The decrease in adrenergic cells produced by T3 was paralleled by a decrease in the number of tyrosine hydroxylase-positive cells, but T3 did not reduce either total or melanocyte cell number. Cultures were sensitive to T3 during the first 5 days in culture and T3 was not cytotoxic to adrenergic cells. The decrease in adrenergic cells seen with T3 was partially reversed by RA suggesting that these two compounds may be working through a common pathway.

BMP-2 and BMP-4, but not BMP-6, increase the number of adrenergic cells which develop in quail trunk neural crest cultures.

The bone morphogenetic proteins (BMPs) are members of the TGF beta superfamily of growth factors. We have investigated the effects of BMP-2, BMP-4, and BMP-6 on the development of quail trunk neural crest cells in tissue culture. The presence of human recombinant BMP-2 or BMP-4 resulted in a dramatic increase in the number of tyrosine hydroxylase (TH)-immunoreactive cells which developed after 7 days in vitro. In contrast, BMP-6 showed little or no effect. Total cell number was not altered significantly by BMP-2, BMP-4, or BMP-6. However, in the presence of BMP-2 and BMP-4, but not BMP-6, the fraction of the cell population that became melanocytes was reduced to about two-thirds the control value. Addition of BMP-2 and BMP-4 also increased the number of Islet-1-immunoreactive and Hu-immunoreactive cells, but the magnitude of these increases was less than that observed for the TH-immunoreactive cell population. The Islet-1-immunoreactive cells were a subset of the TH-immunoreactive cells, while the Hu-positive cells were distinct from the TH-immunoreactive population. Analysis using bromodeoxyuridine labeling indicated that the increased numbers of TH-immunoreactive cells observed in the presence of BMP-2 and BMP-4 were not due to an increased rate of cell division in committed TH precursors. Further experiments, in which cultures were initiated from single cells, showed that the presence of BMP-2 resulted in colonies containing numerous catecholamine-positive cells without altering the overall number of colonies which survived and developed. In summary, our findings indicate that BMP-2 and BMP-4 are potent regulators of the adrenergic development of avian trunk neural crest cells.

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-trans RA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-trans RA and 13-cis RA 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-trans RA 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-alpha, produced an increase in adrenergic cells similar to that seen with all-trans RA. 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-trans RA is mediated by RAR-alpha and possibly RAR-beta. To further define the actions of all-trans RA on the neural crest we incubated cultures with 5-bromo-2'-deoxyuridine (BrdU) to determine whether all-trans RA could affect the rate of proliferation. The results show that while all-trans RA 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-trans RA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.

Persistent correlation between expression of a sulfated carbohydrate antigen and adrenergic differentiation in cultures of quail trunk neural crest cells.

The carbohydrate antigen recognized by monoclonal antibodies such as HNK-1 (first characterized as recognizing human natural killer cells) and NC-1 (raised against quail neural-crest-derived cells) is found on a number of molecules associated with cell differentiation in vertebrates [42]. Previous work has shown that subpopulations of cultured quail trunk neural crest cells can be separated by fluorescence-activated cell sorting (FACS) on the basis of expression of this carbohydrate antigen. When neural crest cells are separated after 2 days in culture, adrenergic cells develop preferentially within the HNK-1-reactive subpopulation [27]. We wished to investigate whether the capacity for adrenergic differentiation remained associated with the HNK-1-positive cell population at later times in vitro, when the percentage of HNK-1-reactive cells has declined. The present study found that neural crest cells separated according to HNK-1-reactivity after 4 days in culture also showed preferential development of adrenergic cells in HNK-1-positive-enriched cultures, indicating that the HNK-1 epitope is persistently expressed in vitro on cells with adrenergic potential after 4 days of culture. To investigate the possible function of this epitope in development of the adrenergic phenotype, HNK-1 was added to unsorted neural crest cell cultures. The presence of antibody resulted in a decrease in the percentage of HNK-1-reactive cells during the initial 24 h after replating, but had no effect on the number of catecholamine-positive cells which developed after 7 days. We conclude that the epitope recognized by the HNK-1 antibody does not appear to function in the induction of the adrenergic phenotype. However, this antigenic determinant is useful as a predictive early marker which defines a subset of neural crest cells that includes those with the ability to undergo adrenergic differentiation.

Number of adrenergic and islet-1 immunoreactive cells is increased in avian trunk neural crest cultures in the presence of human recombinant osteogenic protein-1.

OP-1, also known as BMP-7, is a member of the TGF-beta superfamily of proteins and was originally identified on the basis of its ability to induce new bone formation in vivo. OP-1 mRNA is found in the developing kidney and adrenal gland as well as in some brain regions (Ozkaynak et al. [1991] Biochem. Biophys. Res. Commun. 179:116-123). We have tested the effect of recombinant human OP-1 on quail trunk neural crest cultures. The number of catecholamine-positive cells which developed after 7 days in vitro in the presence of OP-1 was increased in a dose-dependent manner, with a greater than 100-fold maximal stimulation observed. The increase in the number of catecholamine-positive cells in the presence of OP-1 was paralleled by an increase in the number of tyrosine hydroxylase (TH)-positive cells. In contrast, total and melanocyte cell number were unaffected by the presence of OP-1. The number of Islet-1-immunoreactive cells was also increased by OP-1, but to only about half the value seen for TH. Double label experiments revealed these Islet-1-positive cells were a subset of the TH-positive cells. Inhibitors of DNA synthesis prevented the OP-1-mediated increase in adrenergic cell number, indicating that OP-1 does not act on a postmitotic cell population. However, labeling studies with bromodeoxyuridine indicated that OP-1 did not increase the proportion of the cell population engaged in DNA synthesis. Thus, the OP-1-mediated increase in adrenergic cell number most likely occurs as a result of the enhanced survival of a subpopulation of adrenergic precursors or an increase in their probability of adrenergic differentiation, but not by increasing the mitotic rate of adrenergic precursors or adrenergic cells themselves. In contrast to OP-1, TGF-beta 1 decreased adrenergic cell number. When OP-1 and TGF-beta 1 were added simultaneously, TGF-beta 1 antagonized the OP-1-mediated increase in adrenergic cell number in a dose-dependent manner.

Cloning and analysis of a new developmentally regulated member of the basic helix-loop-helix family.

We have isolated a basic helix-loop-helix (bHLH) family member from an embryonic chick-brain cDNA library. This 3.86-Kb cDNA, GbHLH1.4, exhibits extensive sequence similarity in the bHLH domain with Drosophila daughterless and the vertebrate cDNAs E12 and HTF4. Outside of the bHLH region the similarity is significantly reduced. GbHLH1.4 recognizes a 4.0-Kb mRNA and in situ hybridization analysis shows that GbHLH1.4 mRNA is widely expressed at early stages of development but becomes progressively restricted as embryogenesis proceeds. At later stages of embryonic development, mRNA transcripts are localized to several structures including the ventricular layers of the spinal cord and brain, the facial primordia, dorsal root ganglia and heart muscle and cardiac valves. Strikingly, GbHLH1.4 expression in chick embryos exhibits significant overlap with that reported for the murine negative HLH regulator, Id.

Ciliary neurotrophic factor (CNTF) has a dose-dependent biphasic effect on the number of adrenergic cells which develop in avian trunk neural crest cultures.

The embryonic neural crest is the source of progenitor cells for a wide variety of adult cell types including several populations of neurons and neuroendocrine cells. We have investigated the effect of ciliary neurotrophic factor (CNTF) on the development of quail trunk neural crest cultures. We found that at low doses CNTF promoted the development of increased numbers of adrenergic cells, but higher doses resulted in a decreased number of adrenergic cells relative to controls. Total and melanocyte cell number were unaffected over the range of CNTF concentrations tested. These results indicate that CNTF can affect adrenergic development in neural crest cultures in a dose-dependent biphasic manner.

Temporal restriction of neural crest development in vitro: changes in the adrenergic differentiation of neural crest clusters in the presence and absence of an overlay of reconstituted basement membrane-like matrix.

Neural crest clusters were isolated after 18 or 42 h of growth in suspension and plated on a fibronectin substrate in the presence or absence of an overlay of a reconstituted basement membrane-like (RBM) matrix. After 1 week in vitro, cultures of 18-h clusters grown with the RBM gel overlay exhibited a 60-fold increase in the number of catecholamine-positive cells, while the number of melanocytes was decreased to one-quarter the control value. In contrast, clusters isolated after 42 h in suspension developed few adrenergic cells in the presence or absence of the RBM gel overlay. Melanocyte cell number was not altered in 42-h clusters grown in the presence of the RBM gel overlay. Total cell number after 1 week in vitro was similar under all conditions for both 18- and 42-h clusters. The distribution of tyrosine hydroxylase-immunoreactive cells among the experimental conditions was similar to that of catecholamine-positive cells. The presence of the neural tube was not necessary to generate the different developmental properties of clusters isolated at 18 h versus 42 h. These results show that the RBM gel can enhance adrenergic cell development and inhibit melanogenesis in young neural crest cluster cultures. Also, they demonstrate that the capacity of these neural crest cluster cell populations to differentiate and to respond to environmental cues changes as a function of time. These results are compatible either with a model in which the survival of adrenergic precursors within the clusters declines with time or one in which the adrenergic precursors switch their fate to another phenotype.

Temporal expression of HNK-1-reactive sulfoglucuronyl glycolipid in cultured quail trunk neural crest cells: comparison with other developmentally regulated glycolipids.

Monoclonal antibody HNK-1 is an important marker for embryonic neural crest cells and some of their differentiated derivatives. We have identified 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) as one of the HNK-1 antigens present in cultures of trunk neural crest cells. This lipid was present at 2 days in vitro and increased in amount with time in culture. Other major HNK-1-reactive antigens present in the culture were glycoproteins of apparent molecular masses of 120, 180, and 200 kDa. The 180- and 200-kDa bands were present at 2, 7, and 17 days in vitro, whereas the 120-kDa band was present only at 17 days in vitro. Gangliosides GD3, LD1, and LM1 were also found in the cultures and exhibited distinct temporal patterns of expression. Ganglioside GD3 was present at all stages examined and its expression peaked at 7 days in vitro. In contrast, LD1 was present only at 2 days in vitro and was not detectable at later times. Ganglioside LM1 increased in amount with time in culture in a pattern similar to that seen for SGGL-1. Taken together, these results indicate that several HNK-1-reactive molecules are expressed in neural crest cultures in a temporally regulated manner along with several glycolipids that do not bear this epitope.

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.

The phenotypic response of cultured quail trunk neural crest cells to a reconstituted basement membrane-like matrix is specific.

Previous work has demonstrated that a reconstituted basement membrane (RBM)-like matrix stimulates the development of catecholamine (CA)-containing cells in neural crest cultures. In the present work, we found that the proportion of tyrosine hydroxylase and somatostatin immunoreactive cells was increased substantially by an overlay of the RBM matrix. In contrast, there was little or no stimulation of the development of cells possessing several other phenotypic markers including A2B5, E/C8, vasoactive intestinal polypeptide, and the low and middle molecular weight avian neurofilament proteins. These results demonstrate that the response of neural crest cells to the RBM matrix is specific to a small set of phenotypes. In addition, we demonstrate that the phenotype of the adrenergic cells which develop in the presence of the RBM gel overlay is very similar, if not identical, to that of the adrenergic cells which differentiate in the absence of the RBM gel.

Stimulation of adrenergic development in neural crest cultures by a reconstituted basement membrane-like matrix is inhibited by agents that elevate cAMP.

Previous work (Maxwell and Forbes: Development 101:767-776, 1987) has shown that an overlay of reconstituted basement membrane-like (RBM) gel dramatically increased the number of catecholamine-positive (CA+) cells which differentiated in neural crest cultures. We report here that this increase was inhibited when cultures were grown for 7 days in the presence of agents that elevate cAMP, such as 8-bromo-cAMP and 3-isobutyl-1-methylxanthine. The action of 8-bromo-cAMP was dose dependent with a half-maximal effect at about 50 microM. The development of CA+ cells was dramatically reduced when 8-bromo-cAMP was present from days 0-4 in vitro, but was relatively unaffected if 8-bromo-cAMP was present from days 4-7 in vitro. The development of tyrosine hydroxylase immunoreactive cells was also inhibited by 8-bromo-cAMP. The addition of 8-bromo-cAMP increased the number of melanocytes and resulted in either no change or only modest reductions in the number of E/C8 and neurofilament immunoreactive cells, indicating that the effect on CA+ cell ontogeny was selective. In contrast to the effect of 8-bromo-cAMP, addition of 8-bromo-cGMP did not inhibit CA+ cell development in the presence of the RBM gel nor did it stimulate CA+ cell development in the absence of the RBM gel overlay. Our results suggest that cAMP may be an important regulator of phenotypic expression in at least some neural crest cell lineages.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic development of neural crest cells grown in a defined medium under a reconstituted basement-membrane-like matrix.

The embryonic neural crest of vertebrates is the source of a wide variety of adult cell types. We have demonstrated previously that the presence of a reconstituted basement-membrane-like (RBM) gel overlay can dramatically stimulate the development of adrenergic cells in neural crest cultures grown in a complex medium containing horse serum and chick embryo extract. In the present experiments we have analyzed the differentiation of neural crest cells grown in a defined medium with an RBM gel overlay. We found that the presence of the RBM gel promoted the development of catecholamine-containing (CA+) cells in neural crest cultures grown in defined medium compared to cultures grown in this same medium in the absence of the gel. The number of CA+ cells which developed in cultures grown in defined medium in the presence of the RBM gel overlay was similar to that seen in cultures grown in complex medium in the absence of the RBM gel.

Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture.

We have tested the hypothesis that developmentally significant cellular subsets are present in the early stages of neural crest ontogenesis. Cultured quail trunk neural crest cells probed with the monoclonal antibodies HNK-1 and R24 exhibited heterogeneous staining patterns. Fluorescence-activated cell sorting was used to isolate the HNK-1+ and HNK-1- cell populations at 2 days in vitro. When these cell populations were cultured, the HNK-1+ sorted cells differentiated into melanocytes, unpigmented cells, and numerous catecholamine-positive (CA+) cells. In contrast, the HNK-1- sorted cells gave rise to melanocytes and unpigmented cells, but few, if any, CA+ cells. When neural crest cells at 2 days in vitro were labeled with R24 and sorted, both the R24+ the R24- sorted cell populations produced numerous CA+ cell, melanocytes, and unpigmented cells. These results provide evidence for the existence of developmental preferences in some subsets of neural crest cells early in embryogenesis.