Chaya Kalcheim

Formation of the dorsal root ganglia in the avian embryo segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.

In vivo effect of brain-derived neurotrophic factor on the survival of developing dorsal root ganglion cells.

Implantation of silastic membranes between neural tube and somites at somitic levels 20‐24 in 30‐somite‐stage chick embryos results in separation of early migrated neural crest cells of the dorsal root ganglion (DRG) anlage from the neural tube and their death within a few hours [Kalcheim and Le Douarin, (1986) Dev. Biol., 116, 451‐460]. The in vivo effects of brain‐derived neutrotrophic factor (BNDF) on survival of HNK‐1 immunoreactive DRG cells separated from the tube were examined by implantation of laminin‐treated silastic membranes (controls) or BDNF/laminin‐treated membranes. In the presence of BDNF/laminin‐treated membranes, 20/25 grafted embryos fixed 10 h after implantation, contained many rescued cells on the operated side. In contrast, only a few rescued cells on the operated side. In contrast, only a few rescued cells were observed in sections on the operated in 2/11 embryos implanted with laminin‐treated silastic membranes, and no rescued cells at all could be detected in embryos implanted with NGF/laminin‐treated (seven embryos) or untreated silastic membranes (12 embryos). The data presented support the hypothesis that early survival and differentiation of neural crest‐derived sensory cells depend on central nervous system‐derived factor(s). Moreover, this is the first evidence for the in vivo activity of BDNF on survival of developing DRG cells.

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.

Cell lineages in peripheral nervous system ontogeny: medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures.

Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbeccos modified Eagles medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE] or in a chemically defined serum- and CEE-free medium. Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures. Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5-6 days. These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein. In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture. If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died. By sequentially culturing neural crest, first in the wholly synthetic medium for 1-3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells. The majority of these cells and/or their precursors were found to undergo cell division in culture. We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors. Our results reinforce the contention, deduced from in ovo transplantation experiments (see N. M. Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I. Black, Ed.), pp. 3-28. Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.

Brain-derived neurotrophic factor stimulates survival and neuronal differentiation in cultured avian neural crest.

The response of trunk neural crest cells taken from precise levels of the neural axis and cultured together with adjacent somites to Brain-derived neurotrophic factor (BDNF), was examined in cultures grown in a chemically defined medium. In control cultures, the number of neural crest-derived neurons expressing the HNK-1 epitope, increased as a function of somitic level in a caudorostral direction. Treatment of cultures with increasing concentrations of BDNF (50 pg/ml to 1 ng/ml) resulted in a 1.5- to 6-fold stimulation in the number of neurons developing from crest cells excised at advanced and post-migratory stages, whereas early migrating crest cells were responsive only to concentrations equal to or higher than 1 ng/ml of BDNF. Nerve growth factor used at 5 and 30 ng/ml had no effect on survival of HNK-1-positive cells at any of the somitic levels tested. In an attempt to identify the subpopulation of HNK-1-immunoreactive neurons responding to BDNF, control and treated cultures were stained for the HNK-1 antibody in combination with substance P (SP) antibodies (as a marker for sensory neurons). SP immunoreactivity localized to a subpopulation of phase-bright, HNK-1-positive neurons. The absolute number of SP-positive neurons increased 2- to 4-fold upon BDNF treatment; however, their relative proportion within the population expressing the HNK-1 epitope remained essentially unchanged from control to treated cultures (on day 1, 20% as compared to 23.3% and on day 2, 44.6% compared to 49.7% for control and treated cultures, respectively). Taken together, these data suggest that BDNF stimulates primary neuronal differentiation of SP expressing neurons, and/or their survival.

Stimulation of collagen production in vitro by ascorbic acid released from explants of migrating avian neural crest

Embryonic neuronal tissues contain a collagen-stimulating factor, shown to enhance the hydroxylation and secretion of proline-containing macromolecules by cultured muscle cells. Here we report on a similar activity found during avian embryonic development in explants of migrating mesencephalic neural crest. The degree of proline hydroxylation of proteins secreted into the medium was stimulated 2.5-6-fold in neural crest-muscle and neural crest-somite cocultures, as compared with control cultures devoid of crest explants. No such stimulation occurred when cocultures were treated with the enzyme ascorbate oxidase (EC 1.10.3.3), suggesting that the active factor in neural crest explants was ascorbic acid or an ascorbate-like molecule. Further characterization of this molecule was performed in crest explants and other embryonic tissues by using HPLC with amperometric detection: this study revealed that migrating cephalic neural crest contains 1.5 micrograms ascorbic acid per mg protein. Our results suggest that ascorbic acid and/or related molecule(s) could act during development of the nervous system as a trigger for collagen production and subsequent assembly of an extracellular matrix.

The Neural Crest: Subject Index

Foreword Lewis Wolpert Preface N. Le Douarin 1. Methods for identifying neural crest cells and their derivatives 2. The migration of neural crest cells 3. The neural crest: a source of mesenchymal cells 4. From the neural crest to the ganglia of the peripheral nervous system: the sensory ganglia 5. The autonomic nervous system and the endocrine cells of neural crest origin 6. The neural crest: source of pigment cells 7. Cell lineage segregation during neural crest ontogeny Concluding remarks and perspectives Index.

The Neural Crest: Author Index

Foreword Lewis Wolpert Preface N. Le Douarin 1. Methods for identifying neural crest cells and their derivatives 2. The migration of neural crest cells 3. The neural crest: a source of mesenchymal cells 4. From the neural crest to the ganglia of the peripheral nervous system: the sensory ganglia 5. The autonomic nervous system and the endocrine cells of neural crest origin 6. The neural crest: source of pigment cells 7. Cell lineage segregation during neural crest ontogeny Concluding remarks and perspectives Index.