Catherine Ziller

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.

Restrictions of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments

Abstract The behavior of neural crest cells from various origins and of neural crest derivatives were investigated when they were transplanted into a host embryo as supernumerary crest structures. They were inserted into the dorsal trunk between the neural primordium and the somites. In this situation the grafted cells (of quail origin) migrated in the chick host and could be recognized any time after the graft by the structure of their nucleus. After a migration phase, they became exclusively localized in the various sites of arrest of neural crest cells and were found mixed with host crest cells. Their localizations varied according to their origin and their developmental stages at grafting time. Cells of autonomic ganglia (ciliary and sympathetic) had a definitive localization restricted to the autonomic structures of the host. They differentiated into adrenergic or cholinergic cells irrespective of their sympathetic or parasympathetic origin, according exclusively to their localization in the host. They were practically never found in the host dorsal root ganglion (DRG). In contrast, the developmental potentialities of DRG cells are broader and, as far as peripheral nervous system potentialities are concerned, they behave like neural crest cells, i.e., they gave rise to both sensory and autonomic neurones plus adrenomedullary cells. A model for cell line segregation from the neural crest is proposed. Several aspects of this model need further analysis, others are based on well-established experimental data.

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.

A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells

Searching for specific markers of neural crest-derived cell lineages, we immunized mice with glycoproteins purified from adult quail peripheral myelin. We obtained a monoclonal antibody that reacts with myelin and peripheral glial cells. This antibody, to Schwann cell myelin protein (SMP), is specific for the membranes of all Schwann cells, irrespective of whether they are associated with myelinated nerves. SMP persists on Schwann cells in long-term cultures in vitro, but is absent from satellite cells of peripheral ganglia, both in vivo and in vitro. The antigen (a protein doublet of Mr 75,000-80,000) is present in, but not restricted to, the myelin lamellae, since it is distributed along the whole myelinating Schwann cell membrane. In the CNS, SMP appears as a single band of Mr 80,000. SMP is first detectable by immunofluorescence at E6 in the quail, which is at least 6 days earlier than the first appearance of already described markers related to myelination.

Developmental potentialities of cells derived from the truncal neural crest in clonal cultures

The developmental potentialities of single truncal neural crest derived cells were analysed in clonal cultures. The clone-forming ability and differentiation potential of crest cells migrating through the somitic mesoderm of 3-day-old embryos (E3) and of non-neuronal cells of dorsal root ganglia taken at E6-14 were compared. Since most of the cells present in the sclerotomal and rostral parts of the somite at E3 become later on incorporated into the spinal ganglia, one can consider that these two cell populations represent the same derivatives of the trunk neural crest at different developmental stages. After 10 days in vitro, the size of clones and their phenotypic composition varied noticeably, revealing a certain heterogeneity in the founder cell populations in terms of developmental potencies. Clones obtained from migrating neural crest cells at E3 were often large (greater than 1000 cells) and many of them contained neuronal and non-neuronal cells. Dorsal root ganglion cells produced mostly small clones (less than 100 cells) in which only non-neuronal (i.e. glial) phenotypes were expressed. Therefore, both the capacity for proliferation and the differentiation ability of cloned neural crest derived cells decrease considerably with increasing embryonic age. This is even more striking if these results are compared with those obtained previously in our laboratory with single cells cultures of E2 cephalic neural crest. In the latter case, both clone sizes and cellular diversity within the colonies were much higher than with E3 truncal crest and dorsal root ganglia (DRG) non-neuronal cells. The second result of the present work concerns the differentiation of the dormant autonomic neuronal precursors of the DRG. It has been established previously that the non-neuronal cells of the DRG include adrenergic precursors than can differentiate in mass culture of dissociated DRG cells. We show that these cells never differentiate in clonal cultures but depend upon the cell density of the culture. This suggests that cell to cell interaction between crest derived cells are critical in eliciting the differentiation of the adrenergic phenotype.

Plasticity in neural crest cell differentiation

The neural crest is a pluripotent population of cells that are endowed with migratory capacities. It has long been known that the differentiation pathway taken by cells derived from the neural crest is largely controlled by the microenvironment to which they home after their migration phase, indicating a high degree of plasticity in their developmental fate. Recent progress has been made concerning the factors which influence survival, growth and differentiation of selected sets of precursors in each embryonic site colonised by derivatives of the neural crest.

A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

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.

Environmentally Directed Nerve Cell Differentiation: In Vivo and In Vitro Studies

Publisher Summary By using the quail–chick marker system to follow the migration and differentiation of neural crest cells, the neural axis is regionalized at an early stage into “adrenergic” and “cholinergic” sections, from which arise respectively the sympathetic and parasympathetic/enteric ganglioblasts of the autonomic nervous system. This regionalization does not correspond to an irreversible determination of the crest cells because, under certain experimental conditions, cholinergic neurons can develop from the “adrenergic” region of the crest and vice versa. The phenotypic expression of the presumptive ganglion cells appears to be responsive to the environmental conditions they encounter during and/or after their migration. A study of the nature of the factors required to obtain cholinergic and/or adrenergic differentiation has been undertaken by culturing explants of “pure” neural crest from both “cholinergic” and “adrenergic” regions of the neural axis. Results clearly show that the tissue environment in which the neurons develop determines their final phenotype and is responsible for the observed distribution of catecholaminergic cells in the dorsal trunk structures and cholinergic cells in the splanchnopleural derivatives.

In vivo and in vitro expression of vasoactive intestinal polypeptide-like immunoreactivity by neural crest derivatives

Qualitative and quantitative in vivo studies were performed on the development of the neuropeptide vasoactive intestinal polypeptide (VIP) in the peripheral nervous system of quail embryos. VIP-like immunoreactivity (VIPLI) was found by radioimmunoassay (RIA) from the sixth day of embryonic life onward in the sympathetic chain, the esophagus and duodenum, and from day 15 of incubation onward in the adrenal glands and the nodose ganglia. By using immunocytochemistry, we identified cells expressing VIPLI in sensory spinal ganglia of 13- to 15-day-old embryos. In neural crest cultures, cells expressing the VIP phenotype differentiated constantly under various culture conditions, in contrast to other phenotypes which had specific medium requirements, i.e. adrenergic cells or substance P-containing neurons.