Dysregulated TRK signalling is a therapeutic target in CYLD defective tumours.

Individuals with germline mutations in the tumour-suppressor gene CYLD are at high risk of developing disfiguring cutaneous appendageal tumours, the defining tumour being the highly organised cylindroma. Here, we analysed CYLD mutant tumour genomes by array comparative genomic hybridisation and gene expression microarray analysis. CYLD mutant tumours were characterised by an absence of copy-number aberrations apart from LOH chromosome 16q, the genomic location of the CYLD gene. Gene expression profiling of CYLD mutant tumours showed dysregulated tropomyosin kinase (TRK) signalling, with overexpression of TRKB and TRKC in tumours when compared with perilesional skin. Immunohistochemical analysis of a tumour microarray showed strong membranous TRKB and TRKC staining in cylindromas, as well as elevated levels of ERK phosphorylation and BCL2 expression. Membranous TRKC overexpression was also observed in 70% of sporadic BCCs. RNA interference-mediated silencing of TRKB and TRKC, as well as treatment with the small-molecule TRK inhibitor lestaurtinib, reduced colony formation and proliferation in 3D primary cell cultures established from CYLD mutant tumours. These results suggest that TRK inhibition could be used as a strategy to treat tumours with loss of functional CYLD.

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.

Pluripotent neural crest stem cells in the adult hair follicle.

We report the presence of pluripotent neural crest stem cells in the adult mammalian hair follicle. Numerous neural crest cells reside in the outer root sheath from the bulge to the matrix at the base of the follicle. Bulge explants from adult mouse whisker follicles yield migratory neural crest cells, which in clonal culture form colonies consisting of over a thousand cells. Clones contain neurons, smooth muscle cells, rare Schwann cells and melanocytes, demonstrating pluripotency of the clone-forming cell. Targeted differentiation into Schwann cells and chondrocytes was achieved with neuregulin-1 and bone morphogenetic protein-2, respectively. Serial cloning in vitro demonstrated self-renewal capability. Together, the data show that the adult mouse whisker follicle contains pluripotent neural crest stem cells, termed epidermal neural crest cells (eNCSC). eNCSC are promising candidates for diverse cell therapy paradigms because of their high degree of inherent plasticity and due to their easy accessibility in the skin.

Neural crest stem cell and cardiac endothelium defects in the TrkC null mouse.

TrkC null mice have multiple cardiac malformations. Since neural crest cells participate in cardiac outflow tract septation, the aim of this study was to determine at the cellular level the putative neural crest defect. We have identified three types of progenitor cells: stem cells that undergo self-renewal and can generate many cell types, cells that are restricted in their developmental potentials, and cells that are committed to the smooth muscle cell lineage. In TrkC null mice, there is a greater than 50% decrease in stem cell numbers and an equivalent increase in fate-restricted cells. The outflow tract wall is thickened and the endothelial tube is disorganized. We conclude that deletion of the TrkC gene causes precocious fate restrictions of the neural crest stem cell and a defect of the outflow tract endothelium, both of which may contribute to the outflow tract malformations that occur in TrkC null mice.

Ubiquitous embryonic expression of the norepinephrine transporter.

We report that the norepinephrine transporter (NET) is expressed in avian and mouse embryos by numerous tissues that are derived from all three germ layers. In the nervous system, NET is expressed in the neuroepithelium of the brain and the spinal cord (ventral horn and floor plate), forming mesencephalic nuclei, neural crest, dorsal root ganglion, sympathetic ganglion and spinal nerve. Nonneuronal embryonic NET-expressing structures include the olfactory epithelium, the notochord, the somitic dermamyotome and mesenchymal cells in the limb bud. NET is expressed prominently in the cardiovascular system, including endothelial cells of forming blood vessels, the walls of the aorta and veins, the epicardium, myocardium and a subset of blood cells. The gut, lung buds, and in particular the kidneys, are intensely NET immunoreactive. Since neurotransmitters are known to affect proliferation, survival and differentiation of many mesenchymal cell types, NET function may be a physiologically relevant regulatory element in embryonic development. A working model is proposed for neurotransmitter transporter function in the embryo as a system for the concentration and targeted delivery of neurotransmitter.

Differentiating adult hippocampal stem cells into neural crest derivatives.

To investigate the degree of plasticity of hippocampal neural stem cells from adult mice (mHNSC), we have analyzed their differentiation in co-culture with quail neural crest cells. In mixed culture, mHNSC give rise to several non-neuronal neural crest derivatives, including melanocytes, chondrocytes and smooth muscle cells. The data suggest that neural crest cell-derived short-range cues that are recognized across species can instruct adult mHNSC to differentiate into neural crest phenotypes.

Autocrine regulation of norepinephrine transporter expression.

The norepinephrine transporter (NET) is a neurotransmitter scavenger and site of drug action in noradrenergic neurons. The aim of this study was to identify mechanisms that regulate NET expression during the development of quail (q) sympathetic neuroblasts, which develop from neural crest stem cells. Neurotrophin-3 (NT-3) and transforming growth factor beta1 (TGF-beta1) cause an increase of qNET mRNA levels in neural crest cells. When combined, the growth factors are additive in increasing qNET mRNA levels. Both NT-3 and TGF-beta1 are synthesized by neural crest cells. Onset of NET expression precedes the onset of neural crest stem cell emigration from the neural tube. In older embryos, qNET is expressed by several crest-derived and noncrest tissues. The data show that qNET expression in presumptive sympathetic neurons is initiated early in embryonic development by growth factors that are produced by neural crest cells themselves. Moreover, the results support our previous observations that norepinephrine transport contributes to the regulation of the differentiation of neural crest stem cells into sympathetic neurons.

Norepinephrine transporter expression and function in noradrenergic cell differentiation.

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-beta1 (TGF-beta1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.

Factors controlling lineage specification in the neural crest.

The neural crest is a transitory tissue of the vertebrate embryo that originates in the neural folds, populates the embryo, and gives rise to many different cell types and tissues of the adult organism. When neural crest cells initiate their migration, a large fraction of them are still pluripotent, that is, capable of generating progeny that consists of two or more distinct phenotypes. To elucidate the cellular and molecular mechanisms by which neural crest cells become committed to a particular lineage is therefore crucial to the understanding of neural crest development and represents a major challenge in current neural crest research. This chapter discusses selected aspects of neural crest cell differentiation into components of the peripheral nervous system. Topics include sympathetic neurons, the adrenal medulla, primary sensory neurons of the spinal ganglia, some of their mechanoreceptive and proprioceptive end organs, and the enteric nervous system.

Growth factor synergism and antagonism in early neural crest development.

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

Neurotrophin-3- and norepinephrine-mediated adrenergic differentiation and the inhibitory action of desipramine and cocaine.

In the presence of neurotrophin-3 (NT-3), high-affinity norepinephrine (NE) uptake by quail neural crest cells was significantly increased as judged by in vitro colony assay of adrenergic differentiation. In the presence of the related neurotrophins nerve growth factor (NGF) or brain-derived neurotrophic (BDNF) factor, or of basic fibroblast growth factor (bFGF), there were no significant changes. When NE was added to the culture medium in addition to NT-3, more colonies contained dopamine-beta-hydroxylase (DBH)-immunoreactive cells, an enzyme that is characteristic for adrenergic cells. The NE-mediated increase in the portion of colonies that contained DBH-immunoreactive cells was prevented by the tricyclic antidepressant desipramine (DMI) and by cocaine, two types of drug that block cellular transport of NE. To further examine whether NE acts via uptake, colony assays were performed in the presence and absence of adrenergic antagonists and agonists. These would be expected to mimic the DMI and NE effects, respectively, if the mechanism of action involved activation of adrenergic autoreceptors. Neither class of drug showed a detectable effect within a wide range of concentrations. Immunocytochemistry using antibodies against beta 1 and beta 2 adrenergic receptors further supported the notion that DMI action and beta-receptor expression are not causally related. Ratio imaging was subsequently used in an attempt to elucidate the mechanism of NE action. Within a few minutes of addition of NE to the culture medium, there was an increase in intracellular free calcium in a subset of neural crest cells. Taken together, our data indicate that NT-3 is involved in the appearance of the NE transporter (NET) during embryonic development; internalized NE directly or indirectly increases adrenergic differentiation as measured by immunoreactivity of the adrenergic biosynthetic enzyme DBH; and norepinephrine uptake inhibitors have treatogenic potential.

Mitogenic and anti-proliferative signals for neural crest cells and the neurogenic action of TGF-beta1.

The influence of pertinent growth factors on proliferation and differentiation of quail neural crest cell was assessed by in vitro colony assay in a serum-free (0.5% chick embryo-extract supplemented) culture medium. The factors tested included basic fibroblast growth factor (bFGF; FGF-2), neurotrophins, and transforming growth factor-beta-1 (TGF-beta). Both bFGF and neurotrophins are implicated in the development of the peripheral nervous system, whereas TGF-beta can affect cell differentiation and modulate the action of other growth factors. Bromodeoxyuridine (BrdU) incorporation indicated that bFGF is mitogenic to pluripotent neural crest cells (and/or their immediate progeny) and to committed melanogenic cells. However, this was not reflected in an increase in colony size. In contrast, colony size did increase when nerve growth factor (NGF) was present in addition to bFGF. This indicated either that both factors are required to initiate cell proliferation or that at least some bFGF-exposed cells become dependent on neurotrophins for survival. Sequential addition of the factors showed that exposure to bFGF was required prior to the presence of a neurotrophin, thus favoring the latter possibility. All three neurotrophins tested, NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), were capable of supporting survival of pluripotent neural crest cells (or their closely related progeny) in the presence of bFGF. In the absence of bFGF, neurotrophins did not affect colony size. Although the BrdU data indicated that bFGF is also a mitogen for committed melanogenic cells, the size of pigmented colonies did not change in the presence of bFGF alone or of bFGF plus a neurotrophin. This suggested that another, yet to be determined, factor is required for the survival of proliferating melanogenic cells. Colony assays were also performed in the presence and absence of TGF-beta, both alone and in combination with bFGF plus NGF. TGF-beta inhibited proliferation of both pluripotent neural crest cells (and/or their immediate derivatives) and of committed melanogenic cells, causing a decrease in colony size. When TGF-beta was added to the culture medium together with the bFGF/NGF combination, this also caused a significant decrease in colony size, similar to the one observed with TGF-beta alone. TGF-beta blocked proliferation even when the cells were exposed 24 to 48 hr to the bFGF/NGF combination prior to addition of TGF-beta. Neurogenesis increased significantly in the presence of TGF-beta. The number per colony of both adrenergic cells and sensory neuron precursors increased in TGF-beta-treated neuroblast-positive colonies. The following new insights were derived from this study: 1) basic FGF is a mitogen for pluripotent neural crest cells (and/or their immediate derivatives); 2) pluripotent and committed melanogenic neural crest cells that have been exposed to bFGF become dependent on trophic support; 3) all neurotrophins tested (NGF, BDNF or NT-3) can fulfill the trophic requirement of bFGF-exposed pluripotent cells, but not for melanogenic cells; 4) TGF-beta is an anti-proliferative signal for pluripotent neural crest cells and for committed melanogenic cells; 5) the TGF-beta-mediated anti-proliferative signal dominates over the bFGF/neurotrophin-mediated mitogenic signal; and 6) TGF-beta enhances sensory and adrenergic neurogenesis, possibly by acting upon a common neurogenic precursor cell. Furthermore, our work confirms previous reports by other investigators, who showed that bFGF promotes and TGF-beta inhibits proliferation of pigment cells.

Growth factor action in neural crest cell diversification.

At the onset of their migration into the embryo, many neural crest cells are pluripotent in the sense that they have the capacity to generate progeny that consist of more than one cell type. More recently, we have found that there are pluripotent neural crest cell-derived cells even at sites of terminal differentiation. These findings support the notion that cues originating from the microenvironment, at least in part, direct neural crest cell type specification. Based on the rationale that growth factors that are known to support survival of neural crest cell derivatives may have additional functions in progenitor cell development, we have examined the action of pertinent growth factors. Trophic, mitogenic, antiproliferative and differentiation promoting activities were found. Stem cell factor (SCF) is trophic for pluripotent neural crest cells. Contrary to expectation, SCF plus a neurotrophin, rather than SCF alone is trophic for committed melanogenic cells. Basic fibroblast growth factor (bFGF) is mitogenic both for pluripotent cells and committed melanogenic cells. However, the cells become dependent on another factor for survival. Whereas any neurotrophin tested can rescue bFGF-activated pluripotent neural crest cells, the factor that rescues melanogenic cells remains to be determined. Transforming growth factor beta 1 (TGF-beta 1) is a powerful antimitotic signal for all neural crest cells that overrides the bFGF/neurotrophin proliferative signal. Furthermore, SCF promotes differentiation of neural crest cells into cells of the sensory neuron lineage. Neurotrophin-3 (NT-3) specifically promotes high affinity uptake of norepinephrine by neural crest cells and is thus thought to play a critical role in the differentiation of sympathetic neuroblasts. In summary, our data indicate that neurotrophins and other pertinent growth factors affect survival, proliferation and differentiation of neural crest cells at multiple levels and in different lineages. Moreover, our findings emphasise the importance of the concerted action of combinations of growth factors, rather than of individual factors.

Multiple actions of stem cell factor in neural crest cell differentiation in vitro.

The neural crest is a transient tissue of the vertebrate embryo that gives rise to most primary sensory neurons and pigment cells in the adult organism, among other cell types and tissues. Many neural crest cells are pluripotent in the sense that their progeny can generate more than one phenotype. The presence of pluripotent neural crest cell-derived cells at sites of terminal differentiation suggests that location-specific cues from the embryonic environment, such as growth factors, are involved in directing their survival, proliferation, and cell type specification. We have therefore examined the influences of one pertinent growth factor, stem cell factor (SCF), on neural crest cell development by in vitro colony assay in a serum-free culture medium. SCF showed three major effects. (1) SCF is trophic for early neural crest cells, that is, either pluripotent cells and/or their more mature progeny. This effect occurs only if SCF is present throughout the culture period, and it is not observed when a neurotrophin is present in addition to SCF. (2) More colonies contain sensory neuron precursors in the presence of SCF. This effect is neutralized by NGF and neurotrophin-3 (NT-3), but not by brain-derived neurotrophic factor (BDNF). (3) The combination of SCF and any neurotrophin tested (NGF, BDNF, NT-3) is trophic for melanogenic cells, whereas SCF alone does not detectably affect melanogenesis. This suggests either that both types of factor are required for melanotrophic action or that melanogenic cells become dependent on neurotrophins after exposure to SCF. Our observation that SCF is required during the first half of the culture period only, and NGF during the second half only, indicates the latter possibility. Whereas coat color changes in the mouse mutants W (c-kit defect) and Steel (SCF defect) and several in vivo and in vitro studies by other investigators have shown previously that SCF is melanotrophic, they also indicated the requirement of an additional factor, or factors, in melanogenesis. Our data suggest that SCF affects neural crest cell development at multiple levels and that survival of melanogenic cells is mediated by a combination of SCF and a neurotropin, rather than by SCF alone.

In vitro clonal analysis of cardiac outflow tract mesenchyme.

In vitro clonal analyses were performed to gain insight into the mechanisms that control development of neural crest-derived cardiac outflow tract mesenchyme of quail embryos. The cardiac neural crest originates from the posterior rhombencephalic neural tube. The cells leave the neural tube and migrate through the posterior visceral arches to the outflow tract of the heart, where they form the conotruncal ridges. Mesenchyme cells derived from the neural tube from somitic levels 1-3 contained 5 types of clone-forming cells. One class of clones contained up to 6 phenotypes; smooth muscle cells, connective tissue cells, chondrocytes, sensory neuron precursors, serotonergic (putative enteric) neurons, and pigment cells. Another type of clone was totally pigmented, containing melanocytes only, whereas a third type consisted entirely of smooth muscle cells. The remaining classes of clones contained 3 and 4 phenotypes, respectively. Subsequently, mesenchymal cells obtained from posterior visceral arches were cloned in vitro. The major observations from these experiments are the following. 1) The cells have lost the capacity to form sensory neurons. 2) The capacity to form pigment cells is lost as well. 3) Four types of morphologically distinguishable clones were found. The frequency of one type of clone that contains ectomesenchymal cells only (smooth muscle cells, connective tissue cells, and chondrocytes) increased from zero at Hamburger and Hamilton stage 19+/20 to 67% at stage 24, possibly giving rise to visceral arch-derived structures. The frequency of the other 3 types of clones decreased with progressing embryonic development, indicating that these clone-forming cells either pass through the visceral arches, and/or are being converted to cells with fewer developmental potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Pluripotent neural crest cells in the developing skin of the quail embryo.

The neural crest, a migratory population of embryonic cells, gives rise to a wide range of differentiated cell types in the mature vertebrate organism, including the melanocytes of the skin. Little is known about the developmental potentials of neural crest cells toward the end of their migratory phase. We have therefore used in vitro analysis to examine the developmental potential of mesenchymal cells derived from explants of trunk epidermal ectoderm of the quail embryo. Melanocytes which differentiated in the cultures could be identified by their content of melanin granules. To detect different neuronal cells, the cultures were stained with antibodies including anti-dopamine-beta-hydroxylase (anti-DBH), which characterizes sympathoadrenal cells, and AC4, an antibody which recognizes the stage-specific embryonic antigen-1 (SSEA-1) that is expressed by cells in the sensory neuron lineage of the quail embryo, but not by sympathoadrenal cells. Seventy-eight percent of the population of neural crest-derived cells seeding the ectoderm around stage 21 gave rise to colonies containing melanocytes only. Twenty percent, however, generated mixed colonies that contained melanocytes, DBH+ cells, SSEA-1+ cells, and unidentified, unpigmented cells. Small numbers of colonies containing fewer phenotypes were also seen. With increasing embryonic age, the number of colonies containing multiple phenotypes declined, until by stage 30 all neural crest colonies contained melanocytes only. Some colonies had been marked at the single-cell stage, and this provided additional confirmation that each colony-type could be generated from a single cell. Thus the significant finding in this study is that a substantial fraction of the neural crest cells arriving early in the ectoderm are pluripotent cells that are able to give rise to pigment cells, to sympathoadrenal cells, to primary sensory neuron precursors, and possibly to other cells which were not identified here. This observation may have implications for our understanding of the mechanisms that control neural crest cell migration and 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 non-neuronal 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of pluripotent neural crest cells in the embryo and the role of brain-derived neurotrophic factor in the commitment to the primary sensory neuron lineage.

Many early migratory neural crest cells are pluripotent in the sense that their progeny are able to generate more than one differentiated phenotype (Sieber-Blum and Cohen, 1980, Dev. Biol. 80:95-106; Baroffio, Dupin, and Le Douarin, 1988, Proc. Natl. Acad. Sci. USA 85:5325-5329; Bronner-Fraser and Fraser, 1988, Nature 335:161-164; Sieber-Blum, 1989a, Science 243:1608-1611; Ito and Sieber-Blum, 1991, Dev. Biol. 148:95-106). At trunk levels, the neural crest contains two classes (Sieber-Blum and Cohen, 1980) and at posterior rhombencephalic levels, three different classes of pluripotent cells (Ito and Sieber-Blum, 1991). We investigated cell differentiation by in vitro clonal analysis to determine when in development the pool of pluripotent neural crest cells becomes exhausted. The data suggest that different classes of pluripotent cells, precursor cells with more restricted developmental potentials, and apparently committed cells, exist at sites of advanced migration (posterior branchial arches) and even at target sites of neural crest cell differentiation [posterior branchial arches, dorsal root ganglia (DRG), sympathetic ganglia (SG), and epidermal ectoderm]. Some putative classes of pluripotent cells persist well into the second half of embryonic development. These observations have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation. They support the idea that cues originating from the microenvironment affect differentiation of pluripotent neural crest cells. One such signal appears to be brain-derived neurotrophic factor (BDNF). In the presence of BDNF, but not nerve growth factor (NGF), there is a significant increase in the number of neural crest cells per colony that express a sensory neuron-specific marker. Because this increase is not accompanied by a corresponding increase in the total number of cells per colony, this suggests that BDNF plays a role in cell type specification.

Characterization of the norepinephrine uptake system and the role of norepinephrine in the expression of the adrenergic phenotype by quail neural crest cells in clonal culture.

This study investigates the role norepinephrine (NE) may play in regulating the differentiation of quail neural crest cells into sympatho-adrenal cells. Cues originating from the embryonic microenvironment are thought to play an important role during development. It is conceivable that NE has a positive regulatory function because adrenergic expression by quail neural crest cells in clonal culture can be inhibited by NE uptake inhibitors such as desipramine (DMI). This possibility is further supported by the notion that in the avian embryo presumptive adrenergic neural crest cells are likely to encounter catecholamines shortly after they have acquired the NE uptake mechanism. Our present data indicate that neural crest cells in clonal culture express a high affinity NE uptake system that can be inhibited by desipramine. As in the embryo, it appears before noticeable levels of catecholamines are accumulated by neural crest cells, as judged by formaldehyde-induced catecholamine fluorescence (FIF). A comparison of the time course of appearance of different adrenergic markers suggests that immunoreactivity against the biosynthetic enzyme tyrosine hydroxylase (TH) may appear first, and that it is followed very closely by the appearance of detectable levels of dopamine-beta-hydroxylase (DBH) and the NE uptake mechanism. Accumulation of catecholamines (FIF) is observed last. Addition of exogenous NE leads to an increase in adrenergic expression in vitro as judged by an increase in the number of colonies containing FIF-positive cells as well as cells expressing the biosynthetic enzymes TH and DBH. This suggests that exogenous NE can play a positive regulatory role in the differentiation of quail neural crest cells into sympathoadrenal cells.