BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.     

Migrating neural crest cells in the trunk of the avian embryo are multipotent

Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural tube either during their migration or at their sites of localization. Here, we test the developmental potential of migrating trunk neural crest cells by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells as they migrate through the somite. By two days after injection, the LRD-labelled clones contained from 2 to 67 cells, which were distributed unilaterally in all embryos. Most clones were confined to a single segment, though a few contributed to sympathetic ganglia over two segments. A majority of the clones gave rise to cells in multiple neural crest derivatives. Individual migrating neural crest cells gave rise to both sensory and sympathetic neurons (neurofilament-positive), as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells (both neurofilament-negative). Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. Our data demonstrate that migrating trunk neural crest cells can be multipotent, giving rise to cells in multiple neural crest derivatives, and contributing to both neuronal and non-neuronal elements within a given derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual embryonic origin of the mammalian enteric nervous system

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.     

Constitutive expression of preproendothelin in the cardiac neural crest selectively promotes expansion of the adventitia of the great vessels in vivo

Cardiac neural crest cells are essential for normal development of the great vessels and the heart, giving rise to a range of cell types, including both neuronal and non-neuronal adventitial cells and smooth muscle. Endothelin (ET) signaling plays an important role in the development of cardiac neural crest cell lineages, yet the underlying mechanisms that act to control their migration, differentiation, and proliferation remain largely unclear. We examined the expression patterns of the receptor, ET(A), and the ET-specific converting enzyme, ECE-1, in the pharyngeal arches and great vessels of the developing chick embryo. In situ hybridization analysis revealed that, while ET(A) is expressed in the pharyngeal arch mesenchyme, populated by cardiac neural crest cells, ECE-1 expression is localized to the outermost ectodermal cells of the arches and then to the innermost endothelial cells of the great vessels. This dynamic pattern of expression suggests that only a subpopulation of neural crest cells in these regions is responsive to ET signaling at particular developmental time points. To test this, retroviral gene delivery was used to constitutively express preproET-1, a precursor of mature ET-1 ligand, in the cardiac neural crest. This resulted in a selective expansion of the outermost, adventitial cell population in the great vessels. In contrast, neither differentiation nor proliferation of neural crest-derived smooth muscle cells was significantly affected. These results suggest that constitutive expression of exogenous preproET-1 in the cardiac neural crest results in expansion restricted to an adventitial cell population of the developing great vessels.     

A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells.

The Patch (Ph) mutation in mice is a deletion of the gene encoding the platelet-derived growth factor receptor alpha subunit (PDGFR alpha). Patch is a recessive lethal recognized in heterozygotes by its effect on the pattern of neural crest-derived pigment cells, and in homozygous mutant embryos by visible defects in craniofacial structures. Since both pigment cells and craniofacial structures are derived from the neural crest, we have examined the differentiation of other crest cell-derived structures in Ph/Ph mutants to assess which crest cell populations are adversely affected by this mutation. Defects were found in many structures populated by non-neuronal derivatives of cranial crest cells including the thymus, the outflow tract of the heart, cornea, and teeth. In contrast, crest-derived neurons in both the head and trunk appeared normal. The expression pattern of PDGFR alpha mRNA was determined in normal embryos and was compared with the defects present in Ph/Ph embryos. PDGFR alpha mRNA was expressed at high levels in the non-neuronal derivatives of the cranial neural crest but was not detected in the crest cell neuronal derivatives. These results suggest that functional PDGF alpha is required for the normal development of many non-neuronal crest-derived structures but not for the development of crest-derived neuronal structures. Abnormal development of the non-neuronal crest cells in Ph/Ph embryos was also correlated with an increase in the diameter of the proteoglycan-containing granules within the crest cell migratory spaces. This change in matrix structure was observed both before and after crest cells had entered these spaces. Taken together, these observations suggest that functional PDGFR alpha can affect crest development both directly, by acting as a cell growth and/or survival stimulus for populations of non-neurogenic crest cells, and indirectly, by affecting the structure of the matrix environment through which such cells move.     

Identification of early neurogenic cells in the neural crest lineage.

Pluripotent neural crest cells are restricted progressively during development. The sequence of restrictions and the time(s) in early development at which such restrictions are imposed on crest-derived cells are largely unknown. We have used a human autoantibody (Anti-Hu) to characterize neurogenic populations of avian neural crest-derived cells. Anti-Hu binds specifically to neurons and neuroendocrine cells in older (greater than E4) quail embryos. Early in development, Anti-Hu also binds a subpopulation of neural crest-derived cells that lack neuronal morphology and do not express other neuronal traits. These cells may represent a putative neurogenic precursor subpopulation within the early crest cell lineage. To test this hypothesis, we have characterized Anti-Hu immunoreactivity within crest-derived populations known to have, or to lack, the ability to give rise to new neurons. We report that the presence of Anti-Hu+ nonneuronal cells is correlated with the neurogenic ability of a given cell population. Moreover, Anti-Hu+ nonneuronal cells are transient and appear to be replaced by Anti-Hu+ neuronal cells. We conclude that Anti-Hu is a very early indicator of neurogenesis among crest-derived cells and that Anti-Hu+ nonneuronal cells are either neurogenic precursors or immature neurons.     

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.     

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

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

Monoclonal antibodies raised against pre-migratory neural crest reveal population heterogeneity during crest development

In order to address the problem of when heterogeneity arises within premigratory and early migratory neural crest cell populations, mouse monoclonal antibodies were raised against quail premigratory neural crest. Due to the limited availability of immunogen an intrasplenic route for immunization was used. Three monoclonal antibodies (referred to as LH2D4, LH5D3 and LH6C2) were subsequently isolated which recognized subpopulations in 24 h cultures of both quail and chick mesencephalic and trunk neural crest in immunocytochemical studies. Subsequent investigations using a range of six antibodies, including LH2D4, LH5D3 and LH6C2, showed that population heterogeneity (which was not cell cycle related) could be detected as early as 15 h following mesencephalic crest explantation, a stage at which all the neural crest cells were morphologically identical. However, premigratory neural crest from the same axial level of origin was homogeneous, as judged by immunoreactivity patterns with these antibodies. Significant differences were found in the proportion of immunoreactive cells between populations of mesencephalic and trunk neural crest cultures. Double immunofluorescence studies revealed the existence of at least four separate cell populations within individual crest cultures, each identified by their unique antibody reactivity pattern, thus providing some insight into the underlying complexity of subpopulation composition within the neural crest. Immunocytochemical studies on quail embryos from stages 7-22 showed that the epitopes detected by LH2D4, LH5D3 and LH6C2 were not necessarily confined to the neural crest or to cells of crest derivation. All three epitopes displayed a spatiotemporal regulation in their expression during early avian ontogeny. Since the differential epitope expression described in this investigation was detectable as early as 15 h after premigratory neural crest explantation, took place in vitro in the absence of any other cell type and changed progressively with time, we conclude that a certain degree of population heterogeneity can be generated very early in neural crest ontogeny and independently of the tissue interactions that normally ensue in vivo.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Appearance of nerve growth factor receptors on cultured neural crest cells

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.     

Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos.

Transgenic mice carrying the human cytomegalovirus immediate early gene promoter driving the E. coli lacZ gene displayed an unusual cell specific expression of beta-galactosidase during development. LacZ expression was first detected in cells lining the apex of the neural fold of day 8.5 embryos. By day 10 of gestation, expression was prominent in the spinal ganglia, the ganglia of cranial nerves V, VII, VIII, IX, and X, in a line of cells marking the ventrolateral pathway adjacent to the dermamyotome, and in a column of differentiated cells in the entire ventrolateral neural tube posterior to the mesencephalon. Expression was also found in the myotomes. Neural tube explants from day 8.5 embryos cultured in vitro showed lacZ expression in cells migrating away from the explant. We conclude that the HCMV-IEP-lacZ transgene is expressed in a subpopulation of neural crest cells and its early derivatives.     

鸡胚早期神经系统发育中凋亡细胞的分布研究

研究鸡胚18S（Stage）神经系统发育中凋亡细胞的分布及其生物学意义。采用HNK－1和TUNEL免疫组化双染及改变切片方向的方法观察了神经管和神经嵴的凋亡细胞,结果显示：凋亡的细胞在间隔10张横向切片上呈非均匀分布,但在矢状切面凋亡细胞有节段性分布趋势。体节处连续神经嵴没有发现凋亡细胞,而体节与体节之间神经嵴迁离神经管呈游离状并有凋亡细胞,神经管腹侧面体节处间充质细胞呈现细胞凋亡节段性分布。结果表明：鸡胚早期神经系统发育中选择性发生细胞凋亡作用。     

Targeted mutagenesis tools for modelling psychiatric disorders

Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.     

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

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

Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

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.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.