GLIA 4:175-184 (1991)

Glial Cell Lineages in the Neural Crest NICOLE LE DOUARIN, CATHERINE DULAC, ELISABETH DUPIN, AND PATRIZIA CAMERON-CURRY Institut d'Embryologie Cellulaire et Moleculaire du CNRS et du College de France, 94736 Nogent-sur-Marne cedex, France

KEY WORDS

PNS, Schwann cells, differentiation, Avian embryo, Monoclonal antibody

ABSTRACT

We have been studying how and when the different peripheral glial cell lineages individualizeduring avian embryonic development.Three different and complementary experimental approacheswere used for this purpose: 1)the quailkhick chimera system allowed the tracing in uiuo of the origin of the various types of peripheral glial cells (Schwanncells of nerves, satellite glial cells of sensory and autonomicganglia, and enteric glial cells),and the analysis of the non-neuronal cell population of ganglia; 2) characterisation of early cell-type specific markers that discriminate between the different glial cell subpopulations;and 3) analysis of the progeny of neural crest cells in clonal cultures. As a result of these approaches, two novel glial-specific markers, expressed earlier than any previously described myelin components, have been identified and partly characterised. The divergence of glial and neuronal cell lineages is a process that is not completely terminated during the phase of neural crest migration. Whereas some cells are apparently already totally committed to a glial fate at this stage, others retain dual neuronaUglia1 potentialities.

INTRODUCTION It is now generally accepted that the neural crest, a transitory structure of the Vertebrate embryo, is the source of all the glial cells of the peripheral nervous system (PNS)including the peripheral nerves, the sensory, sympathetic, parasympathetic, and enteric ganglia (see Le Douarin, 1982, for a review of the experimental data). We have been interested in our laboratory for several years in trying to understand how the numerous cell lineages arising from the neural crest (Table 1) become segregated in the course of ontogeny. Differentiation of the various types of peripheral glial cells has particularly attracted our attention together with the question of how and when, during the development of the PNS, the glial and neuronal lineages diverge. A case in point concerns the phenotypic variability of the peripheral glia, which fall into three main categories, the satellite cells of the sensory and autonomicganglia, the Schwann cells lining the peripheral nerves, and the enteric glia. In the PNS, a glial sheath covers almost the entire surface of the neuronal cell from its perikaryon, located in ganglia, to the nerve processes. Therefore, with the 01991 Wiley-Liss, Inc.

exception of a few areas, the neuronal cell membrane does not come in direct contact with connective tissue, but only with the inner surface of supporting cells, which thus interacts with the molecular and cellular microenvironment of the neurons. In autonomic and sensory ganglia of the PNS, neuronal cell bodies are enclosed within a capsule of one or more satellite cells and a basal lamina separates each neuron-satellite cell complex from the endoneurial compartment (Pannese 1969, 1974). Interdigitations between neuronal and glial membranes (Pannese 1969) suggest complex relationships between neurons and their supporting cells. Temporal changes in these relationships have been observed in the living mouse by videomicroscopy (Pomeroyand Purves, 1988). Although the satellite glial cells of the peripheral ganglia have been the subject of detailed descriptive studies, only little is known on their molecular properties. Some satellite cells, i.e., in the ciliary ganglion of birds (Hess, 1965) and in the spiral ganglion of several species (for references, see Peters et Address reprint requests to Nicole Le Douarin, Institut d'Embryolo 'e Cellulaire et Moleculaire du CNRS et du College de France. 49 bis. Avenue la Belle Gabriellr, 94736 Nogent-sur-Marnecedex, France

rf6!

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LE DOUARIN ET AL. TABLE 1. Derivatives of the neural crest Classification Peripheral nervous system

Tissue or cell type Neurons

Glial cells

Mesectodermal derivatives

Skeletal tissue

Connective tissue and muscle

Other derivatives

Endocrine and paraendocrine cells

Specific tissues or cells Sensory neurons of Spinal ganglia Trigeminal (V) root gangliona Facial (VII) root ganglion Glossopharyngeal (IX) root (superior) ganglion Vagal (X) root (jugular ganglion) Rohon-Beard cells (amphibians) Autonomic neurons of Sympathetic ganglia Parasympathetic ganglia Enteric ganglia Satellite cells in All sensory ganglia, including geniculate (VII), otic (VIII), petrosal (IX) and nodose (X) Sympathetic ganglia Parasympatheticganglia Enteric ganglia Schwann cells of peripheral nerves Nasal and orbitary skeleton Palate and maxillary skeleton Trabeculaea Sphenoid capsuleb Cranial vault Squamosal Frontalb Otic capsulea Visceral skeleton Dermis, smooth muscle and adipose tissue of the skin in face and ventral part of neck Ciliary muscles Striated muscles of face and neckb Wall of large arteries derived from aortic arches (except endothelial cells) Tooth papillae (except endothelium of blood vessels) Corneal “endothelium” and stromal fibroblasts Meninges in Prosencephalon Mesencephalona Connective tissue of pituitary lacrymal glands salivary glands thyroid parathyroid thymus Dorsal fin mesenchyme (amphibians and fishes) Carotid body type I cells Calcitonin-producing cells (C-cells) Adrenal medulla

Melanocvtes aIn part. %mall contribution.

al., 1976) can produce myelin; however, this is not the rule, since satellite cells are not myelinated in most peripheral ganglia. Axonal processes of peripheral nerves are enclosed within Schwann cell membranes, which are also covered by a basal lamina. They are of two kinds: nonmyelinating Schwann cells, which ensheath groups of small-diameter axons, and myelinating cells, which surround a segment of single large-diameter axon with a multilamellar myelin sheath. The third kind of peripheral glial cell is found in the gut where intramural plexuses of Auerbach and Meissner have a cellular organisation reminiscent in some

respects of that of the CNS (Gershon and Rothman, this volume; Gabella, 1971,1987). In this review we will mainly concentrate on the data resulting from our experiments on the development of neural crest derivatives in the avian embryo. Our approaches to these problems have included the following steps. We first traced in vivo the origin of the various types of peripheral glia by using the quail-chick chimera system; then we undertook a biochemical study of glial cell differentiation. This work, which is still in progress, involves the characterization of cell-type-specificmolecular markers for peripheral glia. Finally, using a culture system which allows cells from the migrating neural

LINEAGES IN THE PERIPHERAL GLIA

177

crest to be cultured singly and to expand clonally in lished data) led to the conclusion that Schwann cells are vitro, we were able to define several types of glial cell exclusively derived from the neural crest. progenitors endowed with more or less restricted developmental potencies.

EMBRYONIC ORIGIN OF THE PERIPHERAL GLIA

DO THE NEURAL CREST-DERIVED NON-NEURONAL CELLS OF THE EMBRYONIC PERIPHERAL GANGLIA CORRESPOND EXCLUSIVELY TO GLIA?

By means of the quail-chick chimera system, in which defined segments of the neural primordium of chick embryos are replaced by their counterparts from a quail (or vice versa) at the same developmental stage, we could establish the fate map of the neural crest (Fig. 1A). In the initial studies, the length of the neural tube segment involved in the experiments corresponded to that of four to six somites (Le Douarin and Teillet, 1973; Le Douarin, 1982). More recent experiments have refined the analysis by grafting the neural tube and associated neural crest corresponding to a single somite (Teillet et al., 1987). In all ganglia of the PNS, with the exception of trigeminal, petrosal, and nodose ganglia, whose neurons have a placodal origin (Fig. 1B) (Narayanan and Narayanan, 1980; Ayer-Le Lievre and Le Douarin, 1982; D’Amico-Martel and Noden, 19831, the neurons and the glial satellite cells of the ganglia, together with the Schwann cells of the peripheral nerves were found to originate from the same area of the neural crest. However, it could not be determined by this technique, in which cell populations and not single cells are involved, whether they derived from common or distinct precursors. Enteric glia, like enteric neurons, were shown to originate from the vagal neural crest, i.e., from the level of the neural axis corresponding to somites 1 to 7. The cells originating from this area reach the gut wall in the pharyngeal region and expand dramatically to colonize the gut down to the cloaca, thus contributing to the complex network of ganglia and nerves of Meissner’s (submucosal) and Auerbach‘s (myenteric) plexuses. An additional, although modest, contribution to the postumbilical gut plexuses is provided by the lumbosacral neural crest, caudal to the level of somite 28. This particular crest region also gives rise to the ganglionated nerve of Remak that runs in the dorsal mesentery from the cloaca up to the biliary ducts. As mentioned above, the Schwann cells lining the peripheral nerves are all generally considered as descending from the neural crest. This statement, however, needs a note of caution since it has been claimed that, at the trunk level, the ventral neural tube itself contributes, although modestly, to the Schwann cells surrounding spinal nerves (Rickmann et al., 1985); in grafts of the ventral part of the quail neural tube into chick embryos, a few quail cells were seen emerging from the ventro-lateral side of the tube (Lunn et al., 1987). However, their fate and their identification as Schwann cells were not demonstrated. Similar experiments realised in our laboratory by M.A. Teillet (unpub-

The cellular composition of the peripheral ganglia includes, besides the neural crest-derived cells, fibroblasts, and endothelial cells of mesodermal origin. The neural crest-derived components are classically considered to differentiate into neurons and glia. However, it has been demonstrated in our laboratory that, at least in the embryo and early postnatal birds, the non-neuronal cell population of all types of PNS ganglia contains resting neuronal precursors able to yield autonomic neurons either of the sympathetic catecholaminergic or of the enteric type. This was shown in a large series of studies in which fragments of quail peripheral ganglia were back-transplanted into the neural crest migration pathway of a younger chick host, either at the adrenomedullary level (somites 18 to 24) (Le Douarin et al., 1978; Le Lievre et al., 1980; Ayer-Le Lievre and Le Douarin, 1982;Schweizer et al., 1983;Dupin, 1984) or at the vagal level of the neural axis (Fontaine-Perus et al., 1988). In this situation, the implanted ganglion becomes dissociated into single cells, among which the non-neuronal cells only are able to survive, while the postmitotic neurons virtually all die. The surviving cells recover the migratory properties of their neural crest ancestors and home to the neural crest-derived structures of the host where they become mixed up with host-derived crest cells. They differentiate into Schwann cells, satellite glial cells, or neurons. Interestingly, the type of neurons formed by these cells corresponds to those found in the ganglion to which they home. Thus, adrenergic cells are found only in sympathetic ganglia, plexuses, and in the adrenal medulla, while in the gut, only nonadrenergic neurons differentiate. A different assay system was used to study the capacity of peripheral ganglion cells to colonize the gut and contribute to the enteric nervous system (ENS). It consisted in the association in vitro or on the chorioallantoic membrane of various types of quail peripheral ganglia with chick aneural postcoecal gut. In this situation the non-neuronal cells of the trunk sensory and sympathetic ganglia showed very little if any capacity to invade the gut, whereas they contain numerous adrenergic precursors (Fontaine-Perus et al. 1988). A different situation is found in cephalic sensory ganglia, in which the non-neuronal cell population is extremely invasive for the gut wall in this assay (Fontaine-Perus et al., 1988) and produces large numbers of enteric neurons and glial cells. This obviously raises the question of whether several types of neuronal and glial precursors can be distinguished in the neural crest-

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LE DOUARIN ET AL.

, PROSENCEPHALON

MESECTODERM

ANTERIOR

RHOMBENCEPHALON

POSTERIOR

I

CERVICAL

SENSORY GANGLIA

S P I N A L CORD S18,

-

THORACIC

s24=

h

SPINAL CORD

S 2 8 Z

SYMPATHETIC GANGLIA

LUMBOSACRAL SPINAL C O R D

A

.s*.:

1B

I

Placadal N E U R O N S

00

Fig. 1. A The fate map of the neural crest established by quailkhick isotopic transplantations and showin the presumptive territories that yield the mesectoderm, the sensory, parasympathetic, and sympatietie ganglia in normal avian development. B: Contribution of placode- and neural crest-derived precursors to the formation of cranial ganglia.

LINEAGES IN THE PERIPHERAL GLIA

derived cell population (see Fontaine-Perus et al., 1988 for a discussion on this question). In nodose, petrose, and geniculate sensory ganglia, neurons and glial cells have different origins. As mentioned before, the neurons are derived from ectodermal placodes and the non-neuronal cells from the neural crest. Nodose ganglia, in which either the neurons or the non-neuronal cells were labelled by the quail marker, were constructed. Such chimeric ganglia were then back-transplanted into a chick host. The progeny of quail cells was analysed and it could be shown that the non-neuronal cells contained,beside glia, neuronal precursors of the sympathetic adrenal (Ayer-LeLievre and Le Douarin, 1982)and of the enteric lineages (FontainePerus et al, 1988). The placode-derived cells did not survive in the host. This demonstrates that crest-derived non-neuronal cells have neuronal as well as glial potentialities, the former being repressed during the normal course of ganglionic development. In contrast, no glial potentiality of placodal cells was discovered by this experimental paradygm. After back transplantations of E4 and E7 quail sensory ganglia into a younger chick host, quail cells differentiated into neurons and satellite cells in the host sympathetic and sensory ganglia and into Schwann cells. In contrast, when ciliary or sympathetic ganglia were similarly back-transplanted, they never gave rise to cells colonising the dorsal root ganglia (DRG) of the host. Only Schwann cells and sympathoblasts, along with their satellite cells, were found in the host (Le Lievre et al., 1980; Schweizer et al., 1983; FontainePerus et al., 1988).This shows that DRG contain precursors of both autonomic neurons and glial (satellite and Schwann)cells. In contrast, even at early developmental stages, autonomic ganglia can give rise after back transplantation to Schwann cells but not to sensory satellite cells and neurons (Le Douarin 1986).This led to the idea that the development of neurons and their satellite cells are closely related and suggested that the neural crest contains common progenitors for neurons and glia (Le Douarin, 1984). In order to further document this point the research of our laboratory was orientated in two directions. One was to find early markers for glial cells. The other was to search in single-cellcultures of crest cells for precursors able to yield glial cells and neurons.

A SEARCH FOR NEW MARKERS OF PERIPHERAL GLIAL CELLS The progressive commitment of neural crest precursors to a glial fate and their differentiation into a particular glial cell type can be followed during development by the synthesis of cell- and stage-specificmarkers. The earliest molecular markers of crest-derived cells such as NCl/HNKl (Abo and Balch, 1981;Vincent et al., 1983; Tucker et al., 1984)or GLNl (Barbu et al., 1986) are not specific for glial cells. Other molecular

179

entities such as the glial fibrillary acidic protein and several myelin constituents (Po, myelin basic protein, and myelin associated glycoprotein) are exclusively glial constituents, but their expression is limited to the terminal steps of glial differentiation. We have defined several molecules appearing sequentially during gliogenesis. From Crest Cell Migration to Gangliogenesis As they migrate, the majority of crest cells exhibit the HNKl epitope, a glucuronic acid-containing carbohydrate present on several unrelated glycolipids and glycoproteins (McGarry et al., 1985; Kruse et al., 1984, 1985; Chou et al., 1985; Shashoua et al., 1986).HNKl immunoreactivity is later acquired by certain nonneural tissues and, in the PNS, glial cells and some neurons continue to express molecules carrying this epitope in the adult. It is noteworthy that the progenitors of mesectodermal derivatives arising in the cephalic neural crest lose HNKl immunoreactivitysoon after they have reached their sites of arrest in the facial and hypobranchial regions of the head (Vincentet al., 1983). Barbu et al. (1986) obtained a monoclonal antibody (Mab) that they named GLNl by immunising a mouse with a homogenate of embryonic quail sensory ganglia. It recognises surface glycoproteins carried by a subpopulation (about 25%)of migratory crest cells and later on by virtually all the glial (satellite and Schwann) cells plus a smaller subpopulation of neurons than that recognised by HNKl. As mentioned above, none of these early crest cell markers shows a strict specificity for the glial cell lineage. Our attempts to define earlier and more specific molecular markers for peripheral glial cells led us to obtain a series of Mabs, two of which, anti-433 and anti-SMP (for Schwann cell myelin protein) (Dulac et al., 1988; Cameron-Curry et al., 1989, 1991), have been analysed in some detail. Both Mabs were obtained by immunising mice with purified myelin glycoproteins from adult quail sciatic nerves and brachial plexuses. The anti-4B3 Mab was retained for further studies because a thorough immunocytochemical analysis of quail embryos from E2 up to hatching showed that expression of the corresponding antigenic determinant was strictly restricted to glial cells (Cameron-Curry et al., 1991).In the PNS, the 4B3 phenotype is expressed at the surface of Schwann cells, satellite cells of sensory and autonomic ganglia, and on enteric glial cells (Fig. 2A). Double labelling of dissociated ganglion cells with anti-4B3Mab and specific neuronal markers (neurofilament proteins antibodies, tetanus toxin binding sites) revealed that neurons do not express the 4B3 phenotype. It is worth noticing that the 4B3 epitope is also present in abundance in the CNS in both the white and grey matter (Fig. 2A). Owing to the complexity of the cellular architecture of CNS, it was not possible to recognize precisely the cell types that expressed the 4B3 antigenic determinant. When cells from brain and spi-

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LE DOUARIN ET AL

Figs. 2,3.

LINEAGES IN THE PERIPHERAL GLIA

nal cord were dissociated, and put in standard culture conditions, expression of this epitope was curiously strongly reduced. It could be shown, however, by using double-labellingimmunofluorescence, that both astrocytes and oligodendrocytes shared 4B3, a finding that correlated well with the pattern of staining observed on CNS sections. The molecular characterisation of the 4B3 epitope revealed that, like HNKl, it consisted of a carbohydrate moiety associated with several, apparently unrelated molecules, some of them also expressing HNKl. During ontogeny, anti-4B3 Mab does not react with neural crest cells during their migratory phase. Anti4B3 immunoreactivityfirst appears at E3.5 t o E4 when the cells begin to aggregate to form ganglia (Fig. 2B) and just before the first appearance of ultrastructurally detectable star-shaped immature satellite cells in ganglia (Pannese 1969,1974). Therefore, the emergence of the 4B3 phenotype could correspond to an early molecular step of glial cell differentiation.

NERVE

181

SPINAL CORD

Mr 9 4 K,

67 K,

4 Fig. 4. Western blot analysis under nonreducing conditions of antiSMP immunoreactivity.A doublet of M, 75,000-80,000is recognised in nerve extracts whereas a singlet of M, 80,000 is found in spinal cord extracts.

Schwann Cell Differentiation and Nerve Myelination Schwann cell precursors migrate along growing s o n s and actively divide during the formation of peripheral nerves. From E5 onward in the quail embryo, Schwann cells lining peripheral nerves start to express the SMP glycoprotein (Dulac et al., 1988). The Western blot technique revealed that SMP migrates in SDS-PAGE under non-reducing conditions as a doublet of M, 75,000-80,000 in the PNS and a single band of M, 80,000 in the CNS (Fig. 4). Expression of SMP was studied in detail because of the strict specificityof its distribution in the PNS at the surface of Schwann cells, irrespective of whether they are associated with myelinated or non-myelinated nerves. SMP is not expressed by satellite cells associated with neuronal cell bodies in sensory and autonomic ganglia nor by enteric glial cells (Fig. 3A).Thus it seems to be the only marker described so far that distinguishes both kinds of Schwann cells from other peripheral glial elements. Furthermore the strict specificity of SMP expression described in situ is also maintained in vitro; SMP is Fig. 2. A: Transverse section of an E8 quail emb o at the trunk level showing anti-4B3 immunoreactivity on spinal Corygrey (g)and white (w)matter, as well as on spinal nerves (n), dorsal root ganglion (d)and sympathetic chain (s). x 72. B: Transverse section of a stage 16 quail embryo at the trunk level. Spinal gan lia and nerves and primary sympathetic chains (arrow) are bright& stained. The ventrolateral part of the spinal cord is also 4B3- ositive. x 226. Fig. 3. A: Transverse section oan! E l 2 quail embryo at the trunk level, showing anti-SMP immunoreactivity only localized on the white matter (w) in spinal cord and on spinal nerves (n). Some intraganglionic fibers are SMP-positive (arrow) in dorsal root gan lion (d) and athetic ganglion (s), but gan lionic neurons and sat3lite cells are %&negative. x 90. B: Two-wee!-old culture of Schwann cells dissociated from sciatic nerves and brachlal plexuses of an E l 3 quail embryo. x 224. C: Neural crest cells after 11days in culture on a feeder layer of mouse 3T3 fibroblasts. x 448.

present in cultured Schwann cells from dissociated sciatic and brachial nerves (Fig. 3B), whereas in cultures of peripheral ganglia, SMP is expressed only by a few cells that we consider as being the Schwann cells lining the intraganglionic fibers. This long-term persistence of SMP expression by cultured Schwann cells in the absence of neurons suggests that the segregation and the stability of the Schwann cell lineage is independent of the presence of neurons. SMP identifies Schwann cells located along axons several days before the onset of myelination, which occurs at Ell-12 in chick embryos, and it also appears in cultures of neural crest cells after 8 days (Fig. 3 0 . SMP expression thus corresponds to an early step in Schwann cell differentiation both in vivo and in vitro. Although SMP can be defined as an early molecular marker of the Schwann cell lineage, its role is still unknown. Characterisation of the gene encoding this molecule is now in progress and will hopefully permit further documentation of its regulation and its role in gliogenesis. In contrast to the first detectable signs of the emergence of the Schwann cell phenotype described above, myelination, the last step of Schwann cell maturation, depends upon axonal signals. The major peripheral myelin glycoprotein, Po, the cDNA of which has been recently cloned in the chick by Barbu (19901, is a membrane glycoprotein of M, 28,000,belonging to the immunoglobulin gene superfamily. Its synthesis is rapidly down-regulatedwhen Schwann cells are put in culture in the absence of myelogenic factors (Poduslo, 1984; Poduslo and Windebank, 1985). Transfection experiments have shown that Po is implicated in adhesion between the different myelin sheaths through homophilic interactions (DUrso et al., 1990; Filbin et al., 1990).

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LE DOUARIN ET AL.

GLIAL CELL PRECURSORS IN THE NEURAL CREST: A CLONAL ANALYSIS IN CULTURE Significant progress has recently been made in replying to the key question of how diverse and how homogeneously distributed are the developmental potentialities of single neural crest cells within the migratory population and when the crest cells aggregate to form ganglia and nerve sheaths. Up to some years ago, the prevailing idea was that neural crest cells in the migratory phase were largely pluripotent, although heterogeneity with respect to their state of determination had not been searched for. Then monoclonal antibody technology helped in defining antigens that were not represented uniformly but were expressed only by subpopulations of crest cells in vivo. This was the case for EC8, a Mab that defines a neuronal marker (Ciment and Weston, 19821, and for the already mentioned GLNl antigen (Barbu et al., 1986). The decisive breakthrough came from clonal cultures of cephalic neural crest cells (Baroffioet al., 1988;Dupin et al., 1990) and from in situ labelling of crest cells by lysinated-rhodamine-dextran (Bronner-Fraser and Fraser, 1988,1989). The two ways mentioned above are complementary rather than redundant, since the culture technique ideally gives information concerning all the potentialities of differentiation that a given cell can express, provided that it finds all the survival and growth factors required to do so in the culture medium. On the other hand, the in vivo labelling experiments reveal what phenotypes are really expressed by individual cells in situ, after the limitations imposed by the embryonic microenvironment. Moreover, colonies can be maintained for several weeks in vitro, while the progressive dilution of the dye through cell divisions makes it perceptible only for a short time. The clonal cultures of crest cells, pioneered several years ago by Cohen and Konisgberg (19751, provided evidence for the existence of differently committed melanocytic and neuronal precursors in the truncal neural crest (Sieber-Blum and Cohen, 1980; Sieber-Blum, 1989) but, in the absence of specific markers, provided no information on the emergence of glial lineages of the PNS. By using a new culture method and a larger battery of phenotypic markers, we have recently ex-

tended the earlier studies. We were able to demonstrate the considerable heterogeneity of migrating cephalic crest cells, with respect to both their proliferation potential and the phenotypes emerging in their progeny. While some neural crest cells are fully committed, able to give rise to a unique cell type (e.g. neurons), most of them in our conditions yield a largely diversified progeny, containing from two to six cell types (Baroffio et al., 1988). In a recent study (Dupin et al., 1990), we undertook a clonal analysis of the neural crest ancestors of glial cells. The Schwann cell phenotype was characterised by its immunoreactivity with both HNKl and anti-SMP Mabs (SMP+ HNKl+ cells) (see the previous section). The in vivo antigenic properties of other peripheral glial cells (i.e., satellite cells of peripheral ganglia and enteric glial cells) led to the assumption that the SMP - HNKl + cells of the clones that did not exhibit any neuronal marker belonged to that category. Unfortunately, we were unable to use the 4B3 antigen as a marker for differentiating glial cells since it is not stably expressed by neural crest cells in vitro. We found that SMP+ Schwann cells differentiated in nearly 90%of the cultures and were distributed in eight categories of clones according to the phenotypes with which they were associated as illustrated in Table 2. The main results of this analysis of 163 clones are as follows. First, 13%of the clonogenic crest cells give rise to a homogeneous population of Schwann cells, implying the existence of a committed Schwann cell progenitor that, interestingly, develops without any interaction with axons. In other clones, SMP- HNKl + putative satellite ganglion cells were present, alone or together with SMP+ HNK1+ cells. Second, the fact that a large category (37%)of the clones contained both neurons and Schwann and non-Schwann glial cells showed that common progenitors for neurons and glia are prevalent in the migrating neural crest. Thirdly, a few cells gave rise to both Schwann cells and melanocytes. Finally, certain clones contained glial cells and neurons together with cartilage, thus showing that, at the migratory stage, the mesectodermal and neural cell lineages are not yet completely segregated. Indeed, one clone was observed in which all the main cell types derived from the neural crest, (i.e., melanocyte, neurons, glia, cartilage) were found. This must have arisen from a cell that can be

TABLE 2. Identification of uarious types of clones according to their phenotypic composition Types of clonesa

Cell types

Schw. Schw. nN non-Schw. non-Schw. nN+N nN melanocytes

{

+

+ nN + cartilage

"Schw.. Schwann cells.

non-Neurons(nN) SMPf SMPHNK1+ HNKIf

N~~~~~~(N) NF+ or TH+ or VIP'

Melanocytes

Cartilage

+ + + + 4-

Percent of clones 13

+ + + + + + +

+

+ +

+

+

34.5 12 37 1.5 0.5 1 0.5

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183

I

TOTIPOTENT CELL

PLURIPOTENT PRECURSORS

UNIPOTENT PROGENITORS

5

cartilage

neurones

\ MESECTODERMAL

adrenergic cells

Schwann cells

satellite glial cells

melanocytes

Y

NEURAL

Fig. 5. Hy othetical model of the generation of NC-derived cell lineages: eac1 full circle indicates a precursor evidenced by clonal experiments described herein. Letters within a circle define developmental potentialities of this precursor. Precursor cells are classified

MELANOCYTIC

LINEAGES

accordingto decreasing numbers of their potentials. Arrows show some p b l e filiations between the different progenitors and are not exaustive. Dashed circles and arrows indicate possible additional precursors, i.e., others than those demonstrated in the present study.

stance, Schwann cells can originate either from a neurono-glial precursor, or from a committed progenitor (i.e.,in the absence of neurons). Later in development,it was found that the non-neuronal cell population of PNS ganglia contains, in addition to glia, silent neuronal precursors, whose differentiation is repressed during normal development,but that can be elicited when their interactions with neurons are experimentally disturbed. This raises the question of how interactions between differentiating glial cells and neurons contribute to stabilize the diverse glial phenotypes. Advances will depend in part on characterizing developmentally regulated molecules that are specific to glial cell types. By using the Mab technology,we have produced new markers of PNS glial cells that are present early in developCONCLUDING REMARKS ment and which thus enable the precise steps in their Although the existence of phenotypically distinct cat- differentiationprocess to be identified. Further characegories of glial cells in the PNS (i.e., myelinating and terization of these molecules and the study of their gene non-myelinatingSchwann cells, satellite ganglion cells, regulation will doubtless help in understanding the and enteric glial cells) has been recognised for many origin and functional maintenance of glial cell diversity. years, little is known about the mechanisms leading to their emergence during ontogeny, or the molecular REFERENCES steps of their maturation. We have presented here evidencethat in the neural crest, precursors of glial cells Abo, T. and Balch, C.M. (1981)A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1).J. Immuof the PNS constitute a very heterogeneous population nol., 127:1024-1029. of cells in different states of determination. For in- Ayer-Le Lievre, C.S. and Le Douarin, N.M. (1982)The early developconsidered as the putative neural crest stem cell (Baroffio et al., unpublished). These results led us to propose a model for cell line segregation during neural crest ontogeny according to which most crest cells are initially multipotent and undergo progressive restrictions of their developmental potentials as they divide during their migration, leading to the arrival of a mixture of differently committed progenitors at the various sites of gangliogenesis (Fig. 5). The presence of differently committed cells endowed with more or less broad pluripotentiality has also been demonstrated by in situ labelling of neural crest cells (Bronner-Fraserand Fraser, 1988,1989).

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