Development 109, 743-752 (1990) 743 Printed in Great Britain ©The Company of Biologists Limited 1990

A widely distributed antigen developmentally regulated in the

OLIVIER POURQUIE, MONIQUE COLTEY, JEAN-LEON THOMAS and NICOLE M. LE DOUARIN

Institut d'embryologie cellulaire el moliculaire du CNRS et du College de France, 49 bis avenue de la Belle Gabnelle, 94 736 Nogent sur Marne, Cedex, France

Summary

We have identified a glycoprotein (BEN) of ively from the fibres. The loss of expression is concomi- 95-100 xK^Mr using a monoclonal antibody. This pro- tant with the onset of intense proliferation of satellite and tein is transiently expressed at the cell surface of the Schwann cells. This modulated expression within the peripherally projecting , i.e. motoneurons of the nervous sytem is unlike that of any surface glycoprotein spinal cord and cranial nuclei, sensory neurons of the so far described in vertebrates. Preliminary biochemical dorsal root and cranial sensory ganglia and sympathetic, analysis indicates that, although it bears the adhesion- parasympathetic and enteric neurons. In vitro cultures associated epitope HNK-1, BEN does not share charac- of dorsal root and sympathetic ganglia have shown that teristics with any previously described axonal glyco- BEN is expressed on neurons but not on glial cells. On protein. Consequently, we speculate that this glyco- motor and sensory neurons, BEN first appears at the protein might be a novel molecule implicated in selective level of the cell body just after withdrawal from the cell adhesion phenomena, such as axonal fasciculation. cycle. Soon afterwards, expression of the antigen ex- tends to the elongating . After a few days, BEN is no longer expressed by the motor and sensory neurons, Key words: monoclonal antibody, BEN, glycoprotein, disappearing first from the cell body and then progress- neurogenesis, HNK-1.

Introduction nants of the epithelial component of the bursa of Fabricius, an organ unique to birds, in which B lympho- Morphogenesis and cell differentiation are processes cytes differentiate. The antigen recognized by this that occur in a highly integrated manner during embry- MAb, which we have named BEN, turned out to be onic development. They are the result of the interplay expressed by several other cell types, including neurons between inherited information and the environmental of both the central (CNS) and peripheral (PNS) ner- influences to which differentiating cells are subjected. vous systems. The pattern of BEN expression in the Cellular interactions are mediated by molecular entities developing nervous system appears to be strictly devel- expressed on the cell surface and interacting either with opmentally regulated, in a way that is suggestive of a defined components of the extracellular matrix (ECM) role for this surface molecule in the establishment of the or with their counterpart on other cells. Deciphering neural network during axonal extension and growth the 'grammar' that underlies cellular interactions during cone navigation. development first necessitates the identification of the molecular structures involved. The search for mono- clonal antibodies (MAb) (Kdhler and Milstein, 1975) specific for certain cell types or defining antigens Materials and methods expressed on certain cell lineages at precise develop- mental stages has been a successful way to approach this Embryos problem. Fertilized eggs from chick {Callus gallus) and quail (Coturnix coturnix japonica) were obtained from commercial sources, In this laboratory, we have long been interested in and were incubated in a rotary incubator at 37 °C. Stages of the development of the nervous and immune systems in development of the embryos were expressed according to which monoclonal antibody technology has revealed Hamburger and Hamilton (1951), in pair of somites for early the existence of several common antigens (Dalchau et stages, or in days of incubation for later stages. al. 1980; Clark et al. 1985; P6ault et al. 1987). We describe here the cellular specificity of a MAb Generation of monoclonal antibodies (MAb) that was prepared in mouse against surface determi- BEN MAb was obtained by the following immunization 744 O. Pourquii and others procedure. The epithelium of the bursa of Fabricius of E15 single bursa homogenized in PBS and mixed with an equal chick embryos was mechanically depleted of hemopoietic volume of incomplete Freund's adjuvant. Four days later, cells, and was then grafted into the spleen of 3-month Balb/C splenocytes were fused with SP2O myeloma cells (Kdhler and mice. One month later, the mice were boosted by direct Milstein, 1975). Hybridoma supernatants were used for im- injection in the spleen (without surgery) of the epithelium of a munocytochemical screening of frozen sections of E15 bursa

Fig. 1. BEN MAb staining of a transverse section of a E14 quail bursa of Fabricius. The follicular (FE) and the luminal (LE) epithelia are strongly immunoreactive. L, lumen; M, mesenchyme. (x90). Fig. 2. In vitro cultures of sympathetic (A) and dorsal root (B) ganglia from E12 chick, grown for 5 days and stained with BEN MAb showing clusters of immunoreactive neurons. (x280). BEN expression in embryonic nervous system 745 of Fabricius, thymus and gut. Various clones presenting a (IBF) column (Corthier et al. 1984). This purified antibody reactivity against the bursal epithelium were obtained. Some was coupled to CNBr-Sepharose (Pharmacia) in order to of them also displayed a reactivity against the ganglionic make the affinity matrix. BEN affinity purification was carried plexuses of the gut. These clones were subcloned twice and out using the batch procedure, with columns made of 10 ml one of them, BEN, was subjected to further immunochemical matrix in 50 ml Falcon tubes. Either frozen E16 bursas or and biochemical examination. The BEN MAb is an IgGl that spinal cords, or whole E5 chicks were used. Tissues were recognizes the bursal epithelium and the enteric plexuses of homogeneized in the extraction buffer (PBS, 0.5% NP40, both chick and quail species. No reactivity was detected on 50 mM phenylmethyl-sulfonide-fluoride (PMSF)) and left on sections of E13 mouse embryos. ice for lh. The homogenate was then centrifuged at 13 000 revs min"1 for at least 3h and the supernatant was Histological and immunocytochemical procedures added to a non-specific column to which affinity-purified Whole embryos (until 8 days), or isolated organs (after 8 mouse IgG (Nordic) had been coupled. The column was left days) were fixed in a solution of 1% acetic acid in 100% to rotate overnight at 4°C, centrifuged and the supernatant ethanol at —20°C, then embedded in paraffin, and sectioned. Immunocytochemical staining was carried out on 5jun serial sections as follows. Paraffin was removed from slides by toluene-ethanol treatment. For immunoperoxidase staining, slides were left 30min in 0.3% H2O2 in phosphate-buffered saline (PBS), and washed 3 times lOmin in PBS containing 5% newborn calf serum (NCS; GIBCO), before application of the BEN MAb. For immunofluorescence, slides were left 30 min in PBS/5 % NCS, before treating with the BEN MAb. The BEN MAb was used either as a culture supernatant, or as 1/500 dilution of ascitic fluid and was applied on slides overnight at 4°C. The slides were then washed 3 times in PBS and the second antibody was applied for 1 h at room tempera- ture. Immunofluorescence staining was performed using either an anti-IgG or an anti-IgGl antibody coupled to fluorescein isothiocyanate (Southern Biotechnologies Associ- ates) and diluted 1/50 in PBS. Immunoperoxidase staining was carried out using an anti-IgGl antibody coupled to horseradish peroxidase (Southern Biotechnologies Associ- ates) and diluted 1/50 in PBS. Slides were then washed three times in PBS and mounted in 90% glycerol and paraphenyle- nediamine (lOOmgr1) in PBS for immunofluorescence; the peroxidase reaction was carried as described by Bucy et al. (1988). We have also used the MAb 13F4 (Rong et al. 1987) as a marker of myogenic lineage; double staining were carried out with the BEN MAb revealed with the peroxidase reac- tion, and 13F4 revealed with the alkaline phosphatase reac- tion as described by Malik and Daymon (1982). Cultures of sympathetic and dorsal root ganglia Cultures of sympathetic ganglia (SG) and dorsal root ganglia (DRG) of E12 chick embryos were obtained by excising the ganglia and dissociating the cells in 0.1% trypsin (Difco), 0.1% EDTA solution in PBS for 15min at 37°C. The cell suspension was then washed with PBS containing 5 % NCS, the cells were plated on 35 mm dishes (Nunc), and grown for 5 days in Dulbecco's Modified Eagle's Medium supplemented with 5% foetal calf serum. Cultures were then fixed in 4% paraformaldehyde, or in 100% ethanol for 1 h, rinsed in PBS and processed for immunostaining. Fig. 3. Immunofluorescence staining with BEN MAb of a Biochemistry transverse section in the cervical region of a stage 16 chick Western blotting was carried out as described elsewhere embryo. A few cells are immunoreactive in the floor plate (Dulac et al. 1988) using E7, E10, E12, E16 and adult DRG, (FPC) and in the ventral zone (VZ) of the neural tube (NT) SG, spinal cord and bursa of Fabricius as well as whole E5 where the postmitotic motoneurons first appear. The arrow embryos. The samples, subjected to SDS-PAGE (Laemmli, points to immunoreactive fibres growing dorsally in the 1970) on 7.5 or 10% acrylamide gels prior to blotting, were neural tube. The double arrow shows the growing ventral homogenized in PBS containing 0.5% Nonidet P40 (NP 40), root exiting into the sclerotome (Scl). (X400). left on ice for 1 h and then centrifuged at 15 000 revs min~l for Fig. 4. Immunoperoxidase staining with BEN MAb of a 15 min in a Sigma microfuge. For the blotting assay, BEN transverse section of a stage 19 chick embryo at the brachial ascitic fluidwa s used at a dilution of 1/250 in washing solution level. At this stage, the ventral roots (VR) are conspicuous, (PBS, 1/1000 Tween 20, 0.25% Bovine Serum Albumin). but no immunoreactive cell could be observed in the DRG. HNK-1 MAb was used as undiluted hybridoma supernatant. Ao, aorta; FPC, floor plate cells; NT, neural tube; VR, The MAb BEN was purified using a DEAE-Trisacryl ventral root; VZ, ventral zone. (xl25). 746 O. Pourquid and others was added to the BEN column, and left to rotate overnight. Besides the bursa, the most striking cell-type-specific The column was then washed five times with the extraction expression of the BEN antigen concerns the myenteric buffer, and the elution step was carried out by adding two plexuses. Strong staining of both Meissner's and Auer- column-volumes of 10 mM diethylamine, pH11.5, and rotating bach's plexuses was first noticed during the screening for lh at 4°C. After centrifugation, the supernatant was procedure and this observation led us to study the collected, dialyzed overnight against water and lyophilized. The purity of the eluate was checked by subjecting an aliquot distribution of the BEN antigen in the developing to PAGE followed by silver staining and Western blotting. nervous system. BEN expression in the embryonic nervous system Results In the embryonic nervous system, BEN was observed transiently on motoneurons of the spinal cord and of Cellular distribution of the BEN antigen during chick motor nuclei of cranial , particularly on the embryo development oculomotor and trochlear nuclei where it was more The initial aim of this study was to find on the bursal precisely examined. It was never found to be expressed epithelium a surface antigen involved in the cellular on of the spinal cord. The neurons of the interactions leading haemopoietic cells to differentiate DRG and of the cranial sensory ganglia also expressed into B lymphocytes, and BEN MAb was isolated BEN transiently. Sympathetic and enteric neurons expressly because of its staining of the epithelium of the show a more durable immunoreactivity with BEN embryonic bursa of Fabricius. Immunoreactivity on the MAb. A study of the expression of BEN during bursal epithelium is first detected in stage 35-36 em- neurogenesis was performed in detail up to stage 37 bryos (E10). As the follicles containing the B lympho- (E10). Immunocytochemistry performed on unfixed cytes develop, the antigen is strongly expressed both by cultures of SG and DRG of E12 chick showed that BEN the epithelium lining the bursal lumen and by the is expressed on the cell membrane (Fig. 2). follicular epithelium (Fig. 1). BEN immunoreactivity persists on the bursal epithelium after birth. BEN expression on spinal motoneurons A transient BEN immunoreactivity was observed on In the nervous system, immunoreactivity with the BEN other tissues, such as the gut epithelium, which is antibody can first be detected on the motoneurons and stained in the duodenal region from stage 21 (E3,5) to the cells of the floor plate (FPC) of stage 16 embryos (28 stage 36 (E10). BEN expression was also detected on somites) (Fig. 3), i.e. soon after these neurons become haematopoietic cells in the stage 28 (E7) spleen and in postmitotic (Hamburger, 1976). Staining can be ob- the adult bone marrow. This aspect of the cellular served in the ventral zone of the neural tube from the specificity of the BEN MAb will be described in detail mesencephalic region to the level of the 18th somite, on in a further report. cell bodies and of the early motoneurons, and on

Fig. 5. (A) Immunofluorescence at the brachial level of a stage 20 chick embryo after immunoreaction with BEN MAb. The region where motoneurons (Mo) differentiate is larger than at stage 19 (cf. Fig. 4), and the first immunoreactive cells in the DRG are visible. (xl25). (B) Section at the brachial level of a stage 21 chick embryo stained by immunoperoxidase after reaction with BEN MAb. The motor and sensory neurons and processes already exhibit a very intense staining. Ao, aorta; DF, dorsal funiculus; DRG, dorsal root ; FPC, floor plate cells; LB, limb bud; NT, neural tube; SG, sympathetic ganglion; SN, spinal ; VR, ventral root. (X75).

sc

VF

Fig. 7. Transverse section of a stage 25 chick embryo at the brachial level. Immunoreaction with BEN MAb, using the peroxidase staining and with 13F4 revealed with alkaline phosphatase. (A) Detail of the ventral zone of the spinal cord (SC) and the (DRG) showing the difference of staining intensity between sensory and motor cell bodies at this stage. (x330). (B) Detail of the spinal nerve (SN) showing the difference of staining between the motor and sensory fibres. FPC, floor plate cells; Mo, motoneurons; MY, myotome; PR, brachial plexus region; SG, sympathetic ganglion; VF, ventral funiculus; VR, ventral root (xl90). BEN expression in embryonic nervous system 747 the FPC. As neurogenesis proceeds, the expression of immunoreactivity has disappeared from the DRG, but the BEN antigen correlates with the craniocaudal the dorsal funiculus and the spinal nerves remain gradient of differentiation of the neural tube. In the stained. At stage 42 (E16), no staining is observed stage 19 embryo (40 somites), immunoreactivity with either in the motoneuron zone or on DRG neurons and the BEN MAb can be detected on the motoneurons and processes. FPC down to the level of the 30th somite (Fig. 4), and in the stage 21 embryo (E3.5), staining is observed on BEN expression in the autonomic nervous system motoneurons and FPC down to the extremity of the Sympathetic chains. In the sympathetic nervous sys- neural tube. By stage 20-21, the intensity and the tem, the first cells to be stained are seen in the primary number of cells stained in the lateral motor columns sympathetic chains at stage 16, soon after aggregation (LMC) has increased, especially at the cranial level of of the sympathoblasts, from the level of the 6th somite the neural tube, and labeled ventral roots composed of down to the level of the 15th somite. In the stage 21 highly fasciculated axons can clearly be observed leav- embryo, the entire length of each primary sympathetic ing the neural tube opposite to the anterior half of the chain is immunoreactive for the BEN MAb (Fig. 5). In sclerotomes (Fig. 5). By stage 23 (E4), when 95% of the stage 26 embryo, aortic and splanchnic plexuses are the motoneurons are born (Hollyday and Hamburger, clearly immunoreactive. Immunoreactivity increases 1977), the staining on the motoneuron cell bodies and with age and is very intense by stage 36-37 (E10) on cell axons is very intense throughout the motor columns and bodies and axons of sympathetic neurons of the para- is at its maximum in the brachial and lumbar regions. vertebral sympathetic chain (Fig. 9), while it has disap- Bifurcations of the ventral root towards the primary peared from spinal motoneurons and DRG. In the late sympathetic chains are clearly visible by stage 23 (E4). These axons correspond to the preganglionic fibres of the visceral neurons of the future columns of Terni, which are not yet segregated from the motoneuron pool. In the brachial and lumbar region, the spinal nerves appear as thick bundles of stained axons, ending in the plexus, where according to Tosney and Land- messer (1985) an accumulation of growth cones exists at that stage. Axons do not penetrate the limb buds before stage 24-25 (E5). A striking feature of BEN expression on the of motoneurons is that it progressively decreases from stage 26 (E5) onwards, while staining of the ventral roots and of the spinal nerves remains bright until stage 28-29 (E6). By stage 30 (E8), when neuromuscular synaptogenesis has already started, no staining can be observed within the muscles and only the main nerve trunks remain faintly stained. By stage 36-37 (E10), the motor columns of the spinal cord and the motor part of the spinal nerves are essentially negative, while the immunoreactivity of the FPC remains conspicuous. BEN expression in spinal ganglia In the DRG, BEN immunoreactivity first appears at stage 19 simultaneously in the first ten ganglia from the 6th down to the 16th somite (Fig. 5). At about stage 21-22, all the DRG of the embryo are immunoreactive, as are the afferences of the sensory neurons forming the dorsal funiculus. Ventrally, the processes follow the motor fibres to form the sensory part of the spinal nerves. By stage 23, the neuronal somas and axons of the DRG are entirely stained (Fig. 6) and, at the level of the spinal nerves, sensory fibres can be distinguished from the motor nerves because of their stronger anti- BEN immunoreactivity; this difference of immunoreac- tivity can be observed until about stage 25-26 (Fig. 7). By stage 32 (E7), BEN expression progressively Fig. 6. Immunofluorescence using BEN MAb on a section decreases and vanishes first from the cell bodies, while of a dorsal root ganglion (DRG) in the brachial region of a the neuronal processes leaving the ganglia to constitute stage 23 chick embryo. Note the very clustered appearance the sensory part of the spinal nerves and the dorsal of the cells, reflecting an epithelium-like structure. DF, funiculus remain stained (Fig. 8). By stage 36 (E10), dorsal funiculus; NT, neural tube. (x440). 748 O. Pourquie' and others

Fig. 8. BEN MAb immunoperoxidase staining of a transverse section at the brachial level of a stage 34-35 (E8) chick embryo. This figure clearly illustrates the disappearance of the expression of the BEN antigen at the level of the motor and sensory cell bodies. In contrast, sympathetic ganglia (SG), spinal nerve (SN), dorsal funiculus (DF) and floor plate cells (FPC) remain strongly immunoreactive. DRG, dorsal root ganglion; VR, ventral root. (x!20). embryo (E16), SG are still heavily stained, whereas in reactive band corresponds to 100 xlO3. When Western the adult, only a weak immunoreactivity remains on blotting was carried out under reducing conditions, no fibres in the SG. signal could be detected with BEN MAb. It appears from affinity purification experiments that, in the spinal Enteric plexuses. Small clusters of neurons of the en- cord and in the bursa of Fabricius, the antibody teric plexuses between oesophagus and gizzard begin to recognizes a single protein, the band observed on silver- exhibit BEN immunoreactivity at stage 21. The number stained gels corresponding exactly to the band visua- of cells and the intensity of the staining subsequently lised by Western blotting (Fig. 11). The apparent mol- increases, but at stage 26 no immunoreactivity can be ecular weight of this protein was not affected by the use detected in the part of the gut posterior to the gizzard. of a reducing agent such as /J-mercaptoethanol (not In the stage 32 embryo (E7), staining becomes detect- shown). able down to the lower part of the gut. Ganglia of the We have also assayed the presence of the glycosyl- plexuses of Meissner and Auerbach are heavily labelled ated HNK-1 epitope on Western blots of immunoaffi- at E16 (Fig. 10) and the staining persists in the adult. nity-purified BEN antigen. It appears that, both in E16 spinal cord and bursa of Fabricius, BEN is immuno- Biochemical characterization of the BEN antigen reactive for MAb HNK-1 (Fig. 11), indicating that this BEN was studied by SDS-PAGE immunoblotting, and antigen is a glycoprotein that belongs to the HNK-1- by immunoaffinity techniques. After Western blotting bearing family. under non-reducing conditions, BEN MAb was found to recognize two molecular forms with different mol- ecular weights according to the organ considered Discussion (Fig. 11): in the spinal cord, SG and DRG, a 95X103 form can be identified, while in the bursa of Fabricius We report here the identification of an antigen whose and in the bone marrow (not shown) the immuno- distribution and biochemical characteristics do not BEN expression in embryonic nervous system 749

DRG CML

?SG,

Fig. 9. Immunofluorescence using BEN MAb on a transverse section of a E10 chick at the thoracic level, showing the different expression of BEN by sympathetic ganglia (SG) and dorsal root ganglia (DRG). SN, spinal nerve. (x250). Fig. 10. Ganglion of the Auerbach's plexus stained with BEN MAb. Transverse section of the proventricle of a E16 chick. CML, circular muscular layer; G, ganglion. (x400). apparently correspond to those of any previously de- and Edelman, 1984; Daniloff et al. 1986), present on scribed molecule and which present a number of longitudinal fascicles of the spinal cord, like the ventral interesting features. fasciculus. Other molecules described in the chick, such as Fll (Rathjen et al. 1987a) and neurofascin (Rathjen et al. 1987ft) also have different expression patterns, The early expression of the antigen occurs in a highly since they are found on interneurons. Apart from LI, segmental manner in the developing nervous system which has been found in other tissues, these molecules, BEN antigen has only been observed on neurons which are transiently present on axonal subsets, are possessing a peripheral projection. Considering the claimed to be specific to the nervous system, whereas early in neurogenesis, the ex- BEN is expressed by a variety of tissues during develop- pression occurs only on neurons whose axons project ment, furthermore all of these antigens have biochemi- out of the neural tube, in a perpendicular direction cal characteristics distinct from those of BEN. The relative to the body axis. Interneurons projecting longi- major nervous system adhesion molecules, N-CAM tudinally in the spinal cord are never stained by the (Rutishauser, 1986; Tosney et al. 1986) and N-cadherin BEN MAb. To our knowledge, a distribution of this (Hatta et al. 1987; Takeichi, 1987), have a wider type has not so far been described in the vertebrate distribution and are expressed on tissues like the nervous system, though several molecules present on neuroepithelium at early stages where BEN is clearly selective axonal subsets during neurogenesis have been absent. Moreover their biochemical properties are also identified (Jessell, 1988). These include TAG-1, which different from BEN ones. is present on early interneurons as their axon extends In the invertebrate nervous system, fasciclins share along the neuroepithelium (Dodd et al, 1988), LI some general characteristics with the BEN antigen. (Rathjen and Schachner, 1984) or NgCAM (Grumet They constitute a group of proteins expressed in a 750 O. Pourquii and others o segmented manner by subsets of growing axons and are O oc o expressed by a variety of other tissues, such as epider- (/) Q (/) CO mis (Patel et al. 1987; Harrelson and Goodman, 1988). Fasciclin III appears to be a homophilic adhesion 3 molecule of Mr 80X10 which, as revealed by the recent cloning of its cDNA, does not belong to any adhesion molecule family so far identified (Snow et al. 1989). It 95—• .100 would be particularly interesting to clone the cDNA encoding BEN to determine whether it might be a vertebrate homologue of this molecule.

Dynamic aspect of the expression of the BEN antigen on motor and sensory neurons An interesting feature of the BEN antigen is its se- quence of expression at the surface of sensory and motor axons. This expression can be divided into two phases that differ in time for motor and sensory axons. In the first phase, there is extensive expression of the antigen on the cell bodies of the newly born neurons of the LMC and the DRG and this corresponds roughly to their withdrawal from the cell cycle. During this phase, immunoreactive neurons are very tightly associated. Labeling lasts from stage 15-16 to stage 28-30 for the LMC, and from stage 20-21 to stage 30 for the DRG. Immunoreactivity first appears according to a cranio- 100 caudal gradient and becomes rapidly predominant in 95- the brachial and lumbar regions. The second phase, which overlaps the first, corresponds to a strong ex- pression of the antigen on the axon fascicles forming the B ventral roots, spinal nerves and the dorsal funiculus. This phase lasts from stage 16 to 25-30 for the motor root, and from stage 21-22 to stage 36-38 for nerve fibres leaving the DRG. Then, at the level of individual neurons, this expression seems to be transferred from the cell body towards the periphery through the devel- oping neurites as though it were associated with the progression of the growth cone to its target. It is striking that positive staining of neurons and axons, in vivo as well as in culture, is always associated with a very tightly clustered state. Moreover, BEN molecules bear the HNK-1 epitope, which is a complex carbohydrate, first described on 100- -95 molecules at the surface of human natural killer cells (Abo and Balch, 1981), on chick neural crest cells and recently on early axons outgrowing from Xenopus neural tube (Nordlander, 1989). HNK-1 epitope has been demonstrated to be associated with adhesion molecules in the nervous system, such as MAG, Fig. 11. (A) Western blot revealed with BEN MAb. N-CAM (Kruse et al. 1984), or with glycolipids, and Extracts of E10 sympathetic ganglia (SG), dorsal root might play a specific role in cell adhesion (Kiinemund et ganglia (DRG), spinal cord (SC), and bursa of Fabricius al. 1988). One can therefore speculate that BEN could (BF) were run on a 10% polyacrylamide gel, the BEN 3 play such a role in the guidance by means of selective molecule corresponds to the 95X10 MT band in the GS, SC, 3 adhesion of CNS and PNS axons when they are growing and DRG lanes and to the lOOxlO in the BF lane. to the periphery. In this respect, it is interesting to note (B) Analysis of the affinity-purified BEN antigen from E16 spinal cord (SC) and bursa of Fabricius (BF): Silver-stained that the disappearance of the antigen corresponds to gel (SS) and Western blot (WB) revealed with BEN MAb. the onset of glial cell proliferation in the DRG (Pan- (C) Western transfer analysis of affinity-purified BEN nese, 1974) and spinal cord (Fujita and Fujita, 1964; antigen from E16 BF and SC using BEN and HNK-1 Fujita, 1965). It also coincides with the onset of inser- MAbs. The small band observed above the BEN antigen is tion and progressive ensheathment of axons by probably due to the binding of the second antibody to Schwann cells, thus disrupting the close axon-axon immunoglobulins leaching from the column. contacts that existed earlier. A role for BEN in neuron- BEN expression in embryonic nervous system 751 neuron and axon-axon adhesion would be in agree- between neuronal membrane vesicles and glial cells is mediated ment with the constant expression observed on enteric by a specific cell adhesion molecule. /. Cell Biol. 98, 1746-1756. HAMBURGER, V. (1976). The developmental history of the motor neurons, even in the adult, where the neurons remain neuron. The F. O. Schmitt Lecture in . closely associated and fibres unmyelinated (Lewellyn- Research Program Bulletin M.I.T. Press, Smith et al. 1983). In this view, the translation of the Cambridge, Mass. expression observed at the level of the individual HAMBURGER, V. AND HAMILTON, H. L. (1951). A series of normal neuron would reflect a gradient in the disruption of stages in the development of the chick embryo. J. Morph. 88, 49-82. interneuronal adhesion from the cell body to the HARRELSON, A. L. AND GOODMAN, C. S. (1988). Growth cone periphery caused by the proliferation of satellite and guidance in insects: Fasciclin II is a member of the Schwann cells, respectively. This hypothesis will be immunoglobulin superfamily. Science 242, 700-707. subjected to experimental scrutiny in in vitro culture of HATTA, K., TAKAGI, S., FUJISAWA, H. AND TAKEICHI, M. (1987). developing neurons and glial cells. Moreover, our Spatial and temporal expression pattern of N-Cadherin cell further studies on the BEN antigen will involve its adhesion molecules correlated with morphogenetic processes of chicken embryos. Devi Biol. 120, 215-227. biochemical and molecular characterization and its HOLLYDAY, M. AND HAMBURGER, V. (1977). An autoradiogTaphic expression on other cell types during ontogeny. study of the formation of the lateral motor column in the chick embryo. Brain Res. 132, 197-208. We thank Jacqueline Giosue" for excellent histological JESSELL, T. M. (1988). Adhesion molecules and the hierarchy of assistance. We also are particularly grateful to Drs Julian neural development. Neuron 1, 8-18. Smith and Christiane Ayer-Le Lievre for their critical reading KOHLER, G. AND MILSTEIN, C. (1975). Continuous culture of fused of the manuscript. We acknowledge Pei Min Rong for helping cells secreting antibody of predetermined specificity. Nature 256, with the double staining experiments using the 13F4 MAb. 495-497. We are also grateful to Yann Rantier and Bernard Henri for KRUSE, J., MAJLHAMMER, R., WERNECKE, H., FAISSNER, A., SOMMER, I., GORIDIS, C. AND SCHACHNER, M. (1984). Neural cell excellent photographic assistance, and to Sophie Gournet for adhesion molecule and Associated Glycoprotein share a artwork. 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