N.M. Le Douarin

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.

Investigations on cell lineage and tissue interactions in the developing bursa of Fabricius through interspecific chimeras

Abstract Embryonic rudiments of the bursa of Fabricius were grafted between quail and chick embryos at various developmental stages. The distinct morphological differences between the nuclei of the quail and those of the chick were used to determine the origin of cells in differentiated tissue at various times long after the initial graft. It was thus possible to demonstrate that the lymphocytes which develop in the bursa of Fabricius do not originate from the epithelium or from the mesenchyme of the rudiment but arise from precursor cells which reach the organ via the circulation. The inflow of hemopoietic stem cells takes place between 7 and 11 days of incubation in the quail embryo. In the chick the influx begins during the 8th day of incubation and extends until at least the 15th day. The basophilic cells which appear in the rudiment during bursal ontogeny were shown to be hemopoietic precursor cells of extrinsic origin. In the early stages of bursal development the basophilic cells seem to stay in the mesenchymal portion of the organ for a while before invading the epithelium. Some of them remain in the mesenchyme and undergo granulocytic differentiation. Only the cells which invade the epithelium develop into lymphocytes. Bursal histogenesis can occur only when the two primary components of the rudiment, i.e., the epithelium and the mesenchyme, are associated. The mesenchyme of the bursa proper cannot be replaced by another kind of mesenchymal substrate for promoting the development of the epithelium. When associated with the somatopleural mesoderm, the latter either remains in an undifferentiated state or even dedifferentiates.

New studies on the neural crest origin of the avian ultimobranchial glandular cells—Interspecific combinations and cytochemical characterization of C cells based on the uptake of biogenic amine precursors

SummaryChick and quail ultimobranchial glandular cells are able to take up l-Dopa from the blood since the 11th day of incubation. They synthesize dopamine and show a faint greenish fluorescence which strongly contrasts during all the embryonic development with the bright fluorescence of carotid body cells. Isotopic and isochronic grafts of neural rhombencephalic primordium from 6 to 10-somite quail embryos are implanted into chick. The the carotid body and the ultimobranchial gland of the host are invaded by quail neurectodermic cells which have respectively the same cytochemical properties than the normal quail UB and CB glandular cells regarding their ability to take up l-Dopa and their subsequent fluorogenic amine content. Quail cells can be identified from host cells by their nuclear characteristics. The present work confirms earlier findings of neural crest origin of UBG cells using the quail-chick marker system associated with electron microscopic characteristics of calcitonin cells.

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.

What is the developmental fate of the neural crest cells which migrate into the pancreas in the avian embryo

Abstract The hypothesis of a neural crest origin for the endocrine cells of the pancreas has been tested in avian embryos. Isotopic and isochronic transplantations of the neural primordium from quail into chick embryos were made at the vagal level of the neural axis (level of somites 1 to 7). Since quail and chick cells can be distinguished by the structure of their nucleus, the migration of the grafted neural crest cells could be followed in the host. Small groups of quail cells were found in the pancreatic tissue apart from both the exocrine and endocrine structures. The latter were characterized cytochemically at various developmental stages in experimental and control embryos by the formol-induced fluorescence technique after l -Dopa injection, and by their affinity for lead hematoxylin. In no cases were the cells originating from the neural crest and showing the quail nuclear marker found with the cytochemical properties of any kind of the pancreatic endocrine cells. In fact, the neural crest cells which migrate into the pancreas differentiate into parasympathetic ganglia, as evidenced by the use of silver impregnation techniques.

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.

THE EXPRESSION PATTERN OF ENDOTHELIN 3 IN THE AVIAN EMBRYO

We investigated the expression pattern of the endothelin 3 gene, of which the mutation, as well as mutation of its receptor (endothelin-B receptor), affects the development of two neural crest derivatives: enteric nervous system and melanocytes. After previous work showing that these neural crest derived cells express endothelin-B receptor or its subtype endothelin-B2 receptor in the avian embryo, we have demonstrated that endothelin 3 is expressed by the environment of enteric nervous system and melanocyte precursors.

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

SummaryThe development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.

The expression patterns of endothelin-A receptor and endothelin 1 in the avian embryo

We investigated the expression pattern of the endothelin-A receptor and endothelin 1 genes, the mutations of which affect the development of the mesectodermal derivatives of the neural crest. We show here that endothelin 1 is expressed by the environment of the cephalic neural crest cells invading branchial arches. Later on, while the neural crest-derived tissues of the head continue to express endothelin-A receptor, endothelin 1 is no longer expressed in their environment.