Elisabeth Dupin

Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium

This article addresses the problem of the segregation of cell lines during the development of peripheral nervous system components from the neural crest. We show here that committed precursors of peripheral neurons are present in the crest before the migration of its cells has started. If cultured in a serum-deprived medium, a subpopulation of the crest cells readily differentiates into neurons without dividing. Neuronal markers such as neurofilament proteins and receptor sites for tetanus toxin are not expressed in the committed neuronal precursors, but appear after a few hours in culture. They are coexpressed in neurons with the mesenchymal intermediate filament protein, vimentin, which is common to all neural crest cells regardless of their prospective fate. A strong inhibitory effect of serum factor(s) on neurite outgrowth is demonstrated. We show also that conditions stimulating proliferation of crest cells are incompatible with promotion of neuronal differentiation and vice-versa.

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.

New Insights into the Development of Neural Crest Derivatives

Publisher Summary This chapter focuses on new insights into the development of neural crest (NC) derivatives. NC is a highly pluripotent structure, the constitutive cells of which while exhibiting a striking migratory behavior spread over an embryo and settle in certain selected areas where they differentiate into a large variety of cell types. By means of the quail or chick-exchange system of definite regions of the neural primordium, prior to the onset of crest-cell migration, a fate map of the NC was constructed. The basic experimental paradigm was to exchange defined territories in an isotopic and isochronic manner between the embryos of two species. The cephalic NC gives rise to a large array of peripheral ganglia. In addition to melanocytes and endocrine cells, the cephalic crest also yields the mesectoderm from which a large majority of the head skeleton originates. The common precursors for glia and neurons are abundant in the NC. The in vitro clonal technique revealed that the cells with the potentiality to give rise to neurons, glia, melanocytes, and ectomesenchyme exist in the migratory cephalic NC, thus showing that the neural, melanocytic, and mesenchymal lineages are not completely segregated at the time of crest-cell dispersion.

1 The Avian Embryo as a Model in Developmental Studies: Chimeras and in Vitro Clonal Analysis

The avian embryo is a model in which techniques of experimental embryology and cellular and molecular biology can converge to address fundamental questions of development biology. The first part of the chapter describes two examples of transplantation and cell labeling experiments performed in ovo. Thanks to the distinctive histologic and immunocytochemical characteristics of quail and chick cells, the migration and development of definite cells are followed in suitably constructed chimeric quail-chick embryos. Isotopic transplantations of neural tube portions between quail and chick, combined with in situ hybridization with a nucleic probe specific for a quail oligodendrocyte marker, allowed study of the origin and migration of oligodendroblasts in the spinal cord. Heterotopic transplantations of rhombomeres were performed to establish the degree of plasticity of these segments of the hindbrain regarding Hox gene expression, which was revealed by labeling with chick-specific nucleic probes. The second part describes in vitro cell cloning experiments devised to investigate cell lineage segregation and diversification during development of the NC. An original cloning procedure and optimal culture conditions permitted analysis of the developmental potentials of individual NC cells taken at definite migration stages. The results revealed a striking heterogeneity of the crest cell population, which appeared to be composed of precursors at different states of determination. Clonal cultures also provide a means to identify subsets of cells that are the target of environmental factors and to understand how extrinsic signals influence the development of responsive cells.

Cell Lineage Studies in Avian Neural Crest Ontogeny

The neural crest of the Vertebrate embryo is a transitory structure arising during the closure of the neural tube and lying on its dorsal aspect. From it originates most of the peripheral nervous system (PNS), including neurons of all the sympathetic, parasympathetic and enteric ganglia and of the majority of sensory ganglia as well as glial cells of all these ganglia. In addition, the neural crest gives rise to Schwann cells of the peripheral nerves and to many other cell types, such as endocrine cells (e. g. adrenomedullary and calcitoninproducing cells), melanocytes and, in the head region, to the mesectoderm (see Le Douarin, 1982, for review).