Abstract
We have identified a glycoprotein (BEN) of 95 –100 ×103Mr using a monoclonal antibody. This protein is transiently expressed at the cell surface of the peripherally projecting neurons, i.e. motoneurons of the spinal cord and cranial nuclei, sensory neurons of the dorsal root and cranial sensory ganglia and sympathetic, parasympathetic and enteric neurons. In vitro cultures of dorsal root and sympathetic ganglia have shown that BEN is expressed on neurons but not on glial cells. On motor and sensory neurons, BEN first appears at the level of the cell body just after withdrawal from the cell cycle. Soon afterwards, expression of the antigen extends to the elongating axon. After a few days, BEN is no longer expressed by the motor and sensory neurons, disappearing first from the cell body and then progressively from the fibres. The loss of expression is concomitant with the onset of intense proliferation of satellite and Schwann cells. This modulated expression within the nervous sytem is unlike that of any surface glycoprotein so far described in vertebrates. Preliminary biochemical analysis indicates that, although it bears the adhesion-associated epitope HNK-1, BEN does not share characteristics with any previously described axonal glycoprotein. Consequently, we speculate that this glycoprotein might be a novel molecule implicated in selective adhesion phenomena, such as axonal fasciculation.
Introduction
Morphogenesis and cell differentiation are processes that occur in a highly integrated manner during embryonic development. They are the result of the interplay between inherited information and the environmental influences to which differentiating cells are subjected. Cellular interactions are mediated by molecular entities expressed on the cell surface and interacting either with defined components of the extracellular matrix (ECM) or with their counterpart on other cells. Deciphering the ‘grammar’ that underlies cellular interactions during development first necessitates the identification of the molecular structures involved. The search for monoclonal antibodies (MAb) (Köhler and Milstein, 1975) specific for certain cell types or defining antigens expressed on certain cell lineages at precise developmental stages has been a successful way to approach this problem.
In this laboratory, we have long been interested in the development of the nervous and immune systems in which monoclonal antibody technology has revealed the existence of several common antigens (Dalchau et al. 1980; Clark et al. 1985; Péault et al. 1987).
We describe here the cellular specificity of a MAb that was prepared in mouse against surface determinants of the epithelial component of the bursa of Fabricius, an organ unique to birds, in which B lymphocytes differentiate. The antigen recognized by this MAb, which we have named BEN, turned out to be expressed by several other cell types, including neurons of both the central (CNS) and peripheral (PNS) nervous systems. The pattern of BEN expression in the developing nervous system appears to be strictly developmentally regulated, in a way that is suggestive of a role for this surface molecule in the establishment of the neural network during axonal extension and growth cone navigation.
Materials and methods
Embryos
Fertilized eggs from chick (Gallus gallus) and quail (Coturnix coturnix japonica) were obtained from commercial sources, and were incubated in a rotary incubator at 37 °C. Stages of development of the embryos were expressed according to Hamburger and Hamilton (1951), in pair of somites for early stages, or in days of incubation for later stages.
Generation of monoclonal antibodies (MAb)
BEN MAb was obtained by the following immunization procedure. The epithelium of the bursa of Fabricius of E15 chick embryos was mechanically depleted of hemopoietic cells, and was then grafted into the spleen of 3-month Balb/C mice. One month later, the mice were boosted by direct injection in the spleen (without surgery) of the epithelium of a single bursa homogenized in PBS and mixed with an equal volume of incomplete Freund’s adjuvant. Four days later, splenocytes were fused with SP2O myeloma cells (Köhler and Milstein, 1975). Hybridoma supernatants were used for immunocytochemical screening of frozen sections of E15 bursa of Fabricius, thymus and gut. Various clones presenting a reactivity against the bursal epithelium were obtained. Some of them also displayed a reactivity against the ganglionic plexuses of the gut. These clones were subcloned twice and one of them, BEN, was subjected to further immunochemical and biochemical examination. The BEN MAb is an IgGl that recognizes the bursal epithelium and the enteric plexuses of both chick and quail species. No reactivity was detected on sections of E13 mouse embryos.
Histological and immunocytochemical procedures Whole embryos (until 8 days), or isolated organs (after 8 days) were fixed in a solution of 1% acetic acid in 100% ethanol at -20°C, then embedded in paraffin, and sectioned. Immunocytochemical staining was carried out on 5 μm serial sections as follows. Paraffin was removed from slides by toluene-ethanol treatment. For immunoperoxidase staining, slides were left 30 min in 0.3% H2O2 in phosphate-buffered saline (PBS), and washed 3 times 10min 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 temperature. Immunofluorescence staining was performed using either an anti-IgG or an anti-IgGl antibody coupled to fluorescein isothiocyanate (Southern Biotechnologies Associates) and diluted 1/50 in PBS. Immunoperoxidase staining was carried out using an anti-IgGl antibody coupled to horseradish peroxidase (Southern Biotechnologies Associates) and diluted 1/50 in PBS. Slides were then washed three times in PBS and mounted in 90% glycerol and paraphenylenediamine (100 mgl-1) in PBS for immunofluorescence; the peroxidase reaction was carried as described by Buoy 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 reaction, and 13F4 revealed with the alkaline phosphatase reaction 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 1h, rinsed in PBS and processed for immunostaining.
Biochemistry
Western blotting was carried out as described elsewhere (Dulac et al. 1988) using E7, E10, E12, E16 and adult DRG, SG, spinal cord and bursa of Fabricius as well as whole E5 embryos. The samples, subjected to SDS-PAGE (Laemmli, 1970) on 7.5 or 10% acrylamide gels prior to blotting, were homogenized in PBS containing 0.5% Nonidet P40 (NP 40), left on ice for 1 h and then centrifuged at 15 000 revs min-1 for 15 min in a Sigma microfuge. For the blotting assay, BEN ascitic fluid was used at a dilution of 1/250 in washing solution (PBS, 1/1000 Tween 20, 0.25% Bovine Serum Albumin). HNK-1 MAb was used as undiluted hybridoma supernatant.
The MAb BEN was purified using a DEAE-Trisacryl (IBF) column (Corthier et al. 1984). This purified antibody was coupled to CNBr-Sepharose (Pharmacia) in order to make the affinity matrix. BEN affinity purification was carried out using the batch procedure, with columns made of 10 ml matrix in 50 ml Falcon tubes. Either frozen E16 bursas or spinal cords, or whole E5 chicks were used. Tissues were homogeneized in the extraction buffer (PBS, 0.5% NP40, 50 mM phenylmethyl-sulfonide-fluoride (PMSF)) and left on ice for 1h. The homogenate was then centrifuged at 13 000 revs min-1 for at least 3h and the supernatant was added to a non-specific column to which affinity-purified mouse IgG (Nordic) had been coupled. The column was left to rotate overnight at 4°C, centrifuged and the supernatant was added to the BEN column, and left to rotate overnight. The column was then washed five times with the extraction buffer, and the elution step was carried out by adding two column-volumes of 10 mM diethylamine, pH 11.5, and rotating for 1h at 4°C. After centrifugation, the supernatant was collected, dialyzed overnight against water and lyophilized. The purity of the eluate was checked by subjecting an aliquot to PAGE followed by silver staining and Western blotting.
Results
Cellular distribution of the BEN antigen during chick embryo development
The initial aim of this study was to find on the bursal epithelium a surface antigen involved in the cellular interactions leading haemopoietic cells to differentiate into B lymphocytes, and BEN MAb was isolated expressly because of its staining of the epithelium of the embryonic bursa of Fabricius. Immunoreactivity on the bursal epithelium is first detected in stage 35 – 36 embryos (E10). As the follicles containing the B lymphocytes develop, the antigen is strongly expressed both by the epithelium lining the bursal lumen and by the follicular epithelium (Fig. 1). BEN immunoreactivity persists on the bursal epithelium after birth.
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. (×90).
A transient BEN immunoreactivity was observed on other tissues, such as the gut epithelium, which is stained in the duodenal region from stage 21 (E3,5) to stage 36 (E10). BEN expression was also detected on haematopoietic cells in the stage 28 (E7) spleen and in the adult bone marrow. This aspect of the cellular specificity of the BEN MAb will be described in detail in a further report.
Besides the bursa, the most striking cell-type-specific expression of the BEN antigen concerns the myenteric plexuses. Strong staining of both Meissner’s and Auerbach’s plexuses was first noticed during the screening procedure and this observation led us to study the distribution of the BEN antigen in the developing nervous system.
BEN expression in the embryonic nervous system
In the embryonic nervous system, BEN was observed transiently on motoneurons of the spinal cord and of motor nuclei of cranial nerves, particularly on the oculomotor and trochlear nuclei where it was more precisely examined. It was never found to be expressed on interneurons of the spinal cord. The neurons of the DRG and of the cranial sensory ganglia also expressed BEN transiently. Sympathetic and enteric neurons show a more durable immunoreactivity with BEN MAb. A study of the expression of BEN during neurogenesis was performed in detail up to stage 37 (E10). Immunocytochemistry performed on unfixed cultures of SG and DRG of E12 chick showed that BEN is expressed on the cell membrane (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. (×280).
BEN expression on spinal motoneurons
In the nervous system, immunoreactivity with the BEN antibody can first be detected on the motoneurons and the cells of the floor plate (FPC) of stage 16 embryos (28 somites) (Fig. 3), i.e. soon after these neurons become postmitotic (Hamburger, 1976). Staining can be observed in the ventral zone of the neural tube from the mesencephalic region to the level of the 18th somite, on cell bodies and axons of the early motoneurons, and on the FPC. As neurogenesis proceeds, the expression of the BEN antigen correlates with the craniocaudal gradient of differentiation of the neural tube. In the stage 19 embryo (40 somites), immunoreactivity with the BEN MAb can be detected on the motoneurons and FPC down to the level of the 30th somite (Fig. 4), and in the stage 21 embryo (E3.5), staining is observed on motoneurons and FPC down to the extremity of the neural tube. By stage 20 –21, the intensity and the number of cells stained in the lateral motor columns (LMC) has increased, especially at the cranial level of the neural tube, and labeled ventral roots composed of highly fasciculated axons can clearly be observed leaving the neural tube opposite to the anterior half of the sclerotomes (Fig. 5). By stage 23 (E4), when 95% of the motoneurons are born (Hollyday and Hamburger, 1977), the staining on the motoneuron cell bodies and axons is very intense throughout the motor columns and is at its maximum in the brachial and lumbar regions. Bifurcations of the ventral root towards the primary 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 Landmesser (1985) an accumulation of growth cones exists at that stage. Axons do not penetrate the limb buds before stage 24 –25 (E5).
Immunofluorescence staining with BEN MAb of a transverse section in the cervical region of a stage 16 chick embryo. A few cells are immunoreactive in the floor plate (FPC) and in the ventral zone (VZ) of the neural tube (NT) where the postmitotic motoneurons first appear. The arrow points to immunoreactive fibres growing dorsally in the neural tube. The double arrow shows the growing ventral root exiting into the sclerotome (Scl). (× 400).
Immunofluorescence staining with BEN MAb of a transverse section in the cervical region of a stage 16 chick embryo. A few cells are immunoreactive in the floor plate (FPC) and in the ventral zone (VZ) of the neural tube (NT) where the postmitotic motoneurons first appear. The arrow points to immunoreactive fibres growing dorsally in the neural tube. The double arrow shows the growing ventral root exiting into the sclerotome (Scl). (× 400).
Immunoperoxidase staining with BEN MAb of a transverse section of a stage 19 chick embryo at the brachial level. At this stage, the ventral roots (VR) are conspicuous, but no immunoreactive cell could be observed in the DRG. Ao, aorta; FPC, floor plate cells; NT, neural tube; VR, ventral root; VZ, ventral zone, (× 125).
Immunoperoxidase staining with BEN MAb of a transverse section of a stage 19 chick embryo at the brachial level. At this stage, the ventral roots (VR) are conspicuous, but no immunoreactive cell could be observed in the DRG. Ao, aorta; FPC, floor plate cells; NT, neural tube; VR, ventral root; VZ, ventral zone, (× 125).
(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. (× l25). (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 ganglion; FPC, floor plate cells; LB, limb bud; NT, neural tube; SG, sympathetic ganglion; SN, spinal nerve; VR, ventral root. (× 75).
(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. (× l25). (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 ganglion; FPC, floor plate cells; LB, limb bud; NT, neural tube; SG, sympathetic ganglion; SN, spinal nerve; VR, ventral root. (× 75).
A striking feature of BEN expression on the soma 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 immunoreactivity can be observed until about stage 25 –26 (Fig. 7).
Immunofluorescence using BEN MAb on a section of a dorsal root ganglion (DRG) in the brachial region of a stage 23 chick embryo. Note the very clustered appearance of the cells, reflecting an epithelium-like structure. DF, dorsal funiculus; NT, neural tube. (×440).
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 dorsal root ganglion (DRG) showing the difference of staining intensity between sensory and motor neuron cell bodies at this stage. (×330). (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 (×l90).
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 dorsal root ganglion (DRG) showing the difference of staining intensity between sensory and motor neuron cell bodies at this stage. (×330). (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 (×l90).
By stage 32 (E7), BEN expression progressively decreases and vanishes first from the cell bodies, while the neuronal processes leaving the ganglia to constitute the sensory part of the spinal nerves and the dorsal funiculus remain stained (Fig. 8). By stage 36 (E10), immunoreactivity has disappeared from the DRG, but the dorsal funiculus and the spinal nerves remain stained. At stage 42 (E16), no staining is observed either in the motoneuron zone or on DRG neurons and processes.
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. (×20).
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. (×20).
BEN expression in the autonomic nervous system Sympathetic chains. In the sympathetic nervous system, the first cells to be stained are seen in the primary sympathetic chains at stage 16, soon after aggregation of the sympathoblasts, from the level of the 6th somite down to the level of the 15th somite. In the stage 21 embryo, the entire length of each primary sympathetic chain is immunoreactive for the BEN MAb (Fig. 5). In the stage 26 embryo, aortic and splanchnic plexuses are clearly immunoreactive. Immunoreactivity increases with age and is very intense by stage 36 –37 (E10) on cell bodies and axons of sympathetic neurons of the paravertebral sympathetic chain (Fig. 9), while it has disappeared from spinal motoneurons and DRG. In the late embryo (E16), SG are still heavily stained, whereas in the adult, only a weak immunoreactivity remains on fibres in the SG.
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. (×250).
Enteric plexuses. Small clusters of neurons of the enteric plexuses between oesophagus and gizzard begin to exhibit BEN immunoreactivity at stage 21. The number of cells and the intensity of the staining subsequently increases, but at stage 26 no immunoreactivity can be detected in the part of the gut posterior to the gizzard. In the stage 32 embryo (E7), staining becomes detectable down to the lower part of the gut. Ganglia of the plexuses of Meissner and Auerbach are heavily labelled at E16 (Fig. 10) and the staining persists in the adult.
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. (×400).
Biochemical characterization of the BEN antigen
BEN was studied by SDS-PAGE immunoblotting, and by immunoaffinity techniques. After Western blotting under non-reducing conditions, BEN MAb was found to recognize two molecular forms with different molecular weights according to the organ considered (Fig. 11): in the spinal cord, SG and DRG, a 95 ×103 form can be identified, while in the bursa of Fabricius and in the bone marrow (not shown) the immunoreactive band corresponds to 100 ×103. When Western blotting was carried out under reducing conditions, no signal could be detected with BEN MAb. It appears from affinity purification experiments that, in the spinal cord and in the bursa of Fabricius, the antibody recognizes a single protein, the band observed on silver-stained gels corresponding exactly to the band visualised by Western blotting (Fig. 11). The apparent molecular weight of this protein was not affected by the use of a reducing agent such as β-mercaptoethanol (not shown).
(A) Western blot revealed with BEN MAb. Extracts of E10 sympathetic ganglia (SG), dorsal root ganglia (DRG), spinal cord (SC), and bursa of Fabricius (BF) were run on a 10% polyacrylamide gel, the BEN molecule corresponds to the 95 × 103Mr band in the GS, SC, and DRG lanes and to the 100 × 103 in the BF lane. (B)Analysis of the affinity-purified BEN antigen from E16 spinal cord (SC) and bursa of Fabricius (BF): Silver-stained gel (SS) and Western blot (WB) revealed with BEN MAb. (C)Western transfer analysis of affinity-purified BEN antigen from E16 BF and SC using BEN and HNK-1 MAbs. The small band observed above the BEN antigen is probably due to the binding of the second antibody to immunoglobulins leaching from the column.
(A) Western blot revealed with BEN MAb. Extracts of E10 sympathetic ganglia (SG), dorsal root ganglia (DRG), spinal cord (SC), and bursa of Fabricius (BF) were run on a 10% polyacrylamide gel, the BEN molecule corresponds to the 95 × 103Mr band in the GS, SC, and DRG lanes and to the 100 × 103 in the BF lane. (B)Analysis of the affinity-purified BEN antigen from E16 spinal cord (SC) and bursa of Fabricius (BF): Silver-stained gel (SS) and Western blot (WB) revealed with BEN MAb. (C)Western transfer analysis of affinity-purified BEN antigen from E16 BF and SC using BEN and HNK-1 MAbs. The small band observed above the BEN antigen is probably due to the binding of the second antibody to immunoglobulins leaching from the column.
We have also assayed the presence of the glycosylated HNK-1 epitope on Western blots of immunoaffinity-purified BEN antigen. It appears that, both in E16 spinal cord and bursa of Fabricius, BEN is immunoreactive for MAb HNK-1 (Fig. 11), indicating that this antigen is a glycoprotein that belongs to the HNK-1-bearing family.
Discussion
We report here the identification of an antigen whose distribution and biochemical characteristics do not apparently correspond to those of any previously described molecule and which present a number of interesting features.
The early expression of the antigen occurs in a highly segmental manner in the developing nervous system BEN antigen has only been observed on neurons possessing a peripheral projection. Considering the central nervous system early in neurogenesis, the expression occurs only on neurons whose axons project out of the neural tube, in a perpendicular direction relative to the body axis. Interneurons projecting longitudinally in the spinal cord are never stained by the BEN MAb. To our knowledge, a distribution of this type has not so far been described in the vertebrate nervous system, though several molecules present on selective axonal subsets during neurogenesis have been identified (Jessell, 1988). These include TAG-1, which is present on early interneurons as their axon extends along the neuroepithelium (Dodd et al, 1988), LI (Rathjen and Schachner, 1984) or NgCAM (Grumet and Edelman, 1984; Daniloff et al. 1986), present on longitudinal fascicles of the spinal cord, like the ventral fasciculus. Other molecules described in the chick, such as F11 (Rathjen et al. 1987a) and neurofascin (Rathjen et al. 1987b) also have different expression patterns, since they are found on interneurons. Apart from LI, which has been found in other tissues, these molecules, which are transiently present on axonal subsets, are claimed to be specific to the nervous system, whereas BEN is expressed by a variety of tissues during development, furthermore all of these antigens have biochemical characteristics distinct from those of BEN. The major nervous system adhesion molecules, N-CAM (Rutishauser, 1986; Tosney et al. 1986) and N-cadherin (Hatta et al. 1987; Takeichi, 1987), have a wider distribution and are expressed on tissues like the neuroepithelium at early stages where BEN is clearly absent. Moreover their biochemical properties are also different from BEN ones.
In the invertebrate nervous system, fasciclins share some general characteristics with the BEN antigen. They constitute a group of proteins expressed in a segmented manner by subsets of growing axons and are expressed by a variety of other tissues, such as epidermis (Patel et al. 1987; Harrelson and Goodman, 1988). Fasciclin III appears to be a homophilic adhesion molecule of Mr 80 ×103 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 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 sequence 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-caudal gradient and becomes rapidly predominant in the brachial and lumbar regions. The second phase, which overlaps the first, corresponds to a strong expression of the antigen on the axon fascicles forming the 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 developing 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 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, N-CAM (Kruse et al. 1984), or with glycolipids, and might play a specific role in cell adhesion (Künemund et al. 1988). One can therefore speculate that BEN could play such a role in the guidance by means of selective adhesion of CNS and PNS axons when they are growing to the periphery. In this respect, it is interesting to note that the disappearance of the antigen corresponds to the onset of glial cell proliferation in the DRG (Pannese, 1974) and spinal cord (Fujita and Fujita, 1964; Fujita, 1965). It also coincides with the onset of insertion and progressive ensheathment of axons by Schwann cells, thus disrupting the close axon-axon contacts that existed earlier. A role for BEN in neuron neuron and axon-axon adhesion would be in agreement with the constant expression observed on enteric neurons, even in the adult, where the neurons remain closely associated and fibres unmyelinated (Lewellyn-Smith et al. 1983). In this view, the translation of the expression observed at the level of the individual neuron would reflect a gradient in the disruption of interneuronal adhesion from the cell body to the periphery caused by the proliferation of satellite and Schwann cells, respectively. This hypothesis will be subjected to experimental scrutiny in in vitro culture of developing neurons and glial cells. Moreover, our further studies on the BEN antigen will involve its biochemical and molecular characterization and its expression on other cell types during ontogeny.
ACKNOWLEDGEMENTS
We thank Jacqueline Giosué for excellent histological assistance. We also are particularly grateful to Drs Julian Smith and Christiane Ayer-Le Lièvre for their critical reading of the manuscript. We acknowledge Pei Min Rong for helping with the double staining experiments using the 13F4 MAb. We are also grateful to Yann Rantier and Bernard Henri for excellent photographic assistance, and to Sophie Gournet for artwork. Financial support was provided by the Centre National de la Recherche Scientifique (CNRS), the Fondation pour la Recherche Médicale Française and the Ligue Française pour la Recherche contre le Cancer. O.P. is a recipient of a fellowship from the CNRS.