Development 109, 105-116(1990)Printed in Great Britain © The Company of Biologists Limited 1990

105

04 and A007-sulfatide antibodies bind to embryonic Schwann cells prior to

the appearance of galactocerebroside; regulation of the antigen by axon-

Schwann cell signals and cyclic AMP

RHONA MIRSKY1, CATHERINE DUBOIS2, LOUISE MORGAN1 and KRISTJAN R. JESSEN1

^Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK2Laboratoire de Biochimie des Antigens, Department of Immunology, Institut Pasteur, Rue du Dr Rowc, 75015 Paris, France

Summary

In the rat sciatic nerve, the relationship betweenSchwann cells, axons, the extracellular matrix andperineurial sheath cells undergoes extensive modifi-cation between embryo day 15 and the onset of myelin-ation during the first postnatal day. Little is knownabout molecular changes in Schwann cells in this import-ant prenatal period.

In the present paper, we use immunofluorescence tostudy the prenatal development and postnatal regulationof the antigen(s) recognized by the 04 monoclonalantibody and a well-characterized rat monoclonal anti-body to sulfatide, A007. We show that, in a series ofimmunochemical tests, the 04 antibody recognizes onlysulfatide in neonatal and adult rat nerves. Both anti-bodies first bind to Schwann cells in the sciatic nerve atembryo day 16—17, and all Schwann cells bind bothantibodies at birth. In the adult nerve, both nonmyelin-forming and myelin-forming cells are labelled with theantibodies. Schwann cells dissociated from embryo day

15 nerves and cultured in the absence of axons developneither 04 nor A007 binding on schedule, and 04-positiveand A007-positive Schwann cells from postnatal nerveslose the ability to bind these antibodies during the firstfew days in culture. Schwann cells in the distal stump oftransected nerves also sharply down-regulate cell sur-face binding of 04. High numbers of 04-positive or A007-positive Schwann cells reappear in cultures treated withagents that mimic or elevate intracellular cAMP. Weconclude that two anti-sulfatide antibodies 04 and A007,recognize an antigen, probably sulfatide, that appearsvery early in Schwann cell development (one to two daysprior to galactocerebroside) but is nevertheless subjectto upregulation by axonal contact or elevation of intra-cellular cAMP.

Key words: Schwann cells, sulfatide, embryo, axons, sciaticnerve, differentiation antigen, 04 antibody.

Introduction

During the development of a peripheral nerve,Schwann cells with flattened sheet-like extensionslocated mainly at the periphery of the primitive nerveare transformed, through proliferation and a series ofmorphological rearrangements with respect to axonsand the extracellular matrix, into the mitotically quiesc-ent non-myelin-forming and myelin Schwann cellsfound in the adult nerve (Peters and Muir, 1959; Diner,1965; Gamble, 1966; Friede and Samorajski, 1968;Webster et al. 1973; Webster and Favilla, 1984; Ziskind-Conhaim, 1988). These two forms of mature Schwanncell are thought to originate from a common precursorcell. In the rat sciatic nerve, the first indication ofdivergent maturation along one of the two adult path-ways is the axonally regulated appearance of galacto-cerebroside on the first Schwann cells induced along themyelin pathway, seen from embryo day 18 onwards in

the rat sciatic nerve (Mirsky et al. 1980; Jessen et al.1985; 19876).

Prior to this time, during the period from embryo day15 to day 18, early Schwann cells in the rat sciatic nerveare known to express the cell surface proteins Ran-l(217c) and A5E3, the cell adhesion molecules Ll/Ng-CAM and N-CAM, laminin and vimentin (Jessen et al.1989; Jessen et al. 1990 and unpublished observations)and NGF receptors (Taniuchi et al. 1986; 1988; Yan andJohnson, 1988). None of these molecules, however,show significant or well-defined changes in expressionduring this early time period that could be used toanalyse the underlying developmental process and theregulatory factors controlling it.

A developmentally regulated lipid antigen defined bythe monoclonal antibody 04 was first described bySommer and Schachner (1981) and was shown to bindto cells of the oligodendrocyte lineage, appearing onthese cells before galactocerebroside (Sommer and

106 R. Mirsky and others

Schachner, 1981; Schwab and Caroni, 1988; Wolswijkand Noble, 1989). It was also present on about 80% ofspindle-shaped Schwann cells in short-term neonatalmouse dorsal root ganglion cultures (Schachner et al.1981) and on 70% -80% of all non-neuronal cells in24 h cultures from chick sympathetic and dorsal rootsensory ganglia throughout the period of developmentfrom embryo day 7 to 16. These 04+ cells were almostcertainly Schwann or satellite cells associated witheither axons or neuronal cell bodies within the ganglia(Rohrer, 1985; Rohrer et al. 1985; Rohrer andThoenen, 1987). The developmental properties of the04 antigen and their distribution on rat Schwann cells insitu or in culture has not previously been described. Ithas been reported, however, that 67% of cells from atransformed rat Schwann cell line, D6P2T, show sur-face labelling with 04 antibodies on growth in serum-containing medium. In these cells, the 04 antibodyreacts with sulfatide in lipid extracts separated on TLCplates, and the antibody reacts with purified sulfatide indot blot experiments (Singh and Pfeiffer, 1985; Bansaland Pfeiffer, 1987). Evidence from studies with poly-clonal antibodies to sulfatide has indicated, however,that sulfatide can be detected on both Schwann cellsand oligodendrocytes only after the first appearance ofgalactocerebroside (Ranscht et al. 1982) and thus it hasremained an open question whether, in some cells, the04 antibody detects another antigen in addition tosulfatide (Bansal et al. 1989).

In this paper, we have shown that the lipid antigen 04is an early Schwann cell differentiation antigen in therat sciatic nerve. We report on its developmentalappearance, its regulation by axonal contact or cAMPand on studies of its chemical identity. In conjunctionwith these studies, we show that another sulfatideantibody, the monoclonal antibody A007, gives similarresults to those obtained with 04 antibodies.

Materials and methods

Tissue preparationCell suspensions of sciatic nerves were prepared fromSprague-Dawley rats. Embryos, 15-19 days old, newborn, 5day and 30 day or older rats were used. The sciatic nerves orcervical sympathetic trunks were excised and where possiblethe epineurial sheath was removed. The tissue was placed inan enzyme mixture containing 2 mg ml"1 collagenase (Worth-ington), 1.25 mg ml"1 trypsin (Gibco) in Dulbecco's ModifiedEagle's Medium (DMEM) without calcium or magnesium,and was then chopped finely. The tissue was incubated at 37°Cand 5 % CO2 for 90min (30 day or older rats), 40min (5 dayold rats), or 20min (newborn and embryo rats). An equalvolume of Minimal Eagle's Medium with 0.02 M HEPESbuffer (MEM-H) containing 10 % calf serum was added andthe tissue gently dissociated through a plastic pipette tip. Thecells were centrifuged for lOmin at 500 g and resuspendedeither in MEM-H with 10% calf serum and antibody, if theywere to be labelled in suspension, or in DMEM with 10 % calfserum or supplemented DMEM (Jessen et al. 1990) with0.1 % calf serum, if they were to be placed in culture.

Cell cultureCell suspensions from sciatic nerves, prepared as above inDMEM with 10% FCS or with modified DMEM containing0.5% calf serum were plated in 50J<1 droplets on to poly-L-lysine-coated glass coverslips, 13 mm in diameter, in a 24-wellmultiwell plate and kept at 37°C in an incubator gassed with5% CO2/95% air. After 3h, the cultures were topped upwith 400 j/J DMEM containing 10% FCS or with sup-plemented DMEM containing 0.5 % calf serum and main-tained for various time periods. In some experiments 10~5 M-cytosine arabinoside was added for 48 h, 16 h after plating, toeliminate fibroblastic cells (Brockes et al. 1979). Cyclic AMPanalogues, cholera toxin (ISOngml"1) or forskolin (2/JM)were added to some cultures from embryo day 15 or newbornsciatic nerves. The embryo day 15 cells were cultured in thepresence of irradiated 3T3 cells in fully defined medium and0.1% calf serum. Forskolin was added at 24h and thenreplaced at 72 h. Newborn Schwann cells were cultured for 5days in DMEM plus 10 % calf serum, after which the mediumwas changed to DMEM plus 0.1% calf serum. Cyclic AMPanalogues, 5X10~4M 8-bromocyclic AMP plus 5X10~4Mdibutyryl cAMP were added at day 5 and replaced by5xlO~ M doses of each analogue at days 6 and 7, choleratoxin was added for lh at days 5 and 6, and forskolin wasadded at day 5 and replaced on days 6 and 7. Cells fromcultures of both ages were labelled with 04 and 007 antibodies4 days after first addition of the cAMP analogues, choleratoxin or forskolin. Dissociated cell cultures were preparedfrom dorsal root ganglia (DRG) of 8 day rats essentially asdescribed previously (Mirsky et al. 1986). The culture mediaused were the same as those described above for sciatic nervecultures except that NGF (lOOngml"') was added as asupplement to the medium.

DenervationAdult Sprague-Dawley rats, weighing 90-100 g, were anaes-thetized and the left sciatic nerve cut 2—4 mm below the sciaticnotch and a 2-3 mm segment of the nerve removed. Theproximal stump was ligated and rerouted into an adjacentmuscle. After surgery, rats were kept for up to 2 monthsbefore the nerves were removed for dissociation and analysis.

AntibodiesSupernatant fluid containing mouse monoclonal antibody 04(Sommer and Schachner, 1981) was used routinely at adilution of 1:1. It is of the IgM subclass. Rabbit antiserum tobovine S-100 protein (DAKO Immunoglobulins) was used ata dilution of 1:400 in dried and fixed preparations, and at1:800 on cells in dissociated cultures. Mouse monoclonalantibody 217c, in the form of culture supernatant, was used ata dilution of 1:500. This antibody, first described by Peng et al.(1982) has been shown by Fields and Dammerman (1985) toshow extensive similarities with the anti-Ran-1 originallydescribed by Fields et al. (1975). Rat monoclonal antibodyA007 in the form of culture supernatant, was used at a dilutionof 1:1. It is of the IgM subclass and has been well character-ized using TLC and ELISA assays and is essentially specificfor sulfatide (K. Stefansson, personal communication).Monoclonal antibody to galactocerebroside (Ranscht et al.1982) in the form of ascites fluid was used at a dilution of1:200. Rabbit antiserum to N-CAM (Gennarini et al. 1984)was used at a dilution of 1:500. Rabbit antiserum to galacto-cerebroside (Hirayama et al. 1984) was used at a dilution of1:100. Tetramethyl rhodamine conjugated to goat anti-mouseIg (G anti-MIg-Rd) (Cappel Labs.), absorbed with rabbit Igto remove cross-reacting antibodies, was used at a dilution of

Prenatal Schwann cell development 107

1:100. Fluorescein conjugated to goat anti-rabbit Ig (G-anti-R]g-FI) absorbed with mouse Ig to remove cross-reactingantibodies was used at a dilution of 1:100. Tetramethylrhodamine conjugated to goat anti-rat Ig(G anti-Ratlg-Rd)(Nordic Ltd) was used at a dilution of 1:100. Donkey anti-rabbit ]g or anti-rat Ig, biotinylated and streptavidin-fluor-escein or streptavidin-Texas Red (Amersham Internationalpic) were used at a dilution of 1:100. Control antibodies, anirrelevant monoclonal IgM for 04 or IgG^ for 217c, andnormal rabbit serum for the rabbit antibodies, were used atappropriate dilutions.

ImmunofluorescenceAntibodies were diluted as described previously (Jessen et al.19876) and all incubations carried out for 30min at roomtemperature.

Cell suspensionsThese were immunolabelled with 04 or 217c antibodies byadding the antibody to cells suspended in MEM-H with 10%calf serum. After 30 min cells were washed and incubated withG-anti-Mlg-Rd for 30 min, then dried on to gelatin-coatedslides. In experiments where cells were double labelled with04 antibodies and rabbit antiserum to galactocerebroside,cells were labelled sequentially with 04 antibodies,G-anti-MIg-Rd, rabbit anti-galactocerebroside andG-anti-RIg-Fl prior to drying on to microscope slides. Cellsthat were to be double labelled with 04 and S-100 antibodieswere first labelled in suspension with 04 antibodies andG-anti-Mlg-Rd, then dried on to slides, rehydrated on theslide for 20 min in 4% paraformaldehyde in phosphate-buffered saline (PBS), washed, then permeabilized for 10minin 95% ethanol/5% acetic acid. After thorough washing,cells were sequentially labelled with S-100 antibodies, bio-tinylated donkey anti-rabbit Ig followed by streptavidin-fluorescein.

Cultured cellsThese were incubated with 04 antibodies followed byG-anti-MIg-Rd, fixed in 4% paraformaldehyde for 20 min,followed by 95% ethanol/5% acetic acid for 10min. Asimilar procedure was followed with the rat monoclonal A007except that G-anti-Ratlg-Rd was used as a second layer oralternatively biotinylated donkey anti-rat Ig followed bystreptavidin-Texas Red. The cells were then incubated inS-100 antibody followed by G-anti-RIg-Fl. In some exper-iments, 04 labelling was followed by sequential labelling withrabbit anti-galactocerebroside antibodies followed byG-anti-RIg-Fl. In this case, cells were postfixed for 10min in4% paraformaldehyde in PBS. In some experiments, embry-onic cells were cultured for 2h prior to labelling with 04 orA007 antibodies. In this case, cells were fixed for 10 min in4% paraformaldehyde prior to immunolabelling with 04 orA007 antibodies, subsequent steps in the procedure beingidentical to those described above.

Teased nerve preparationsSciatic nerves from adult rats were desheathed, fixed in 4%paraformaldehyde in PBS for 20 min, rinsed in PBS and gentlyteased out on to a polylysine-coated microscope slide in adrop of PBS, using 23-gauge needles as described previously(Jessen and Mirsky, 1984). They were allowed to dry forseveral hours before application of 04 antibodies, followed byG-anti-MIg-Rd or rat A007 antibodies, followed byG-anti-Ratlg-Rd.

Fibres were then labelled with antibodies to N-CAM

followed by G-anti-RJg-Fl. Preparations were post-fixed for10 min in 4% paraformaldehyde in PBS.

All preparations were mounted in Citifluor anti-fade mount-ing medium and viewed for immunofluorescence with a Zeissmicroscope using x63 oil-immersion or x40 dry phase con-trast lenses, epi-illumination and rhodamine and fluoresceinoptics.

Lipid extraction and identificationSciatic nerves were excised from adult and newborn rats andimmediately frozen on dry ice. Lipids were extracted from thenerves using chloroform: methanol:H2O (5:10:3 by vol) andseparated into upper and lower phases using Folch partition(partition 1) as described previously (Dubois et al. 1986;Rougon et al. 1986).

Lipids from the upper phase were desalted on a SephadexG-50 column (20x1.2cm) in H2O then lyophilized. Lipidsfrom the lower phase were submitted to alkali treatment tohydrolyse both neutral lipids and phospholipids using 0.3 MKOH in methanol:H2O (95:5 by vol) for 2h at 37 °C. Afterdesalting on a Sephadex G25 (Pharmacia) column in chloro-form: methanol: H2O (60:30:4.5 by vol), lipids were passedthrough a Unisil (Clarkson Chemical Co. Inc) 100-200 meshcolumn (10x0.6cm) in chloroform. Hydrophobic lipids werefirst eluted with 10 column volumes of chloroform. Then,more polar lipids were eluted with 6 column volumes of twodifferent chloroform-methanol mixtures (4:1 and 1:1 by vol)successively. Lipids from both upper and lower phases werethen separated into neutral and acidic lipids, respectively, byrunning on a column of DEAE-Sephadex in the acetate form(6x0.6cm). The neutral glycolipids were eluted with chloro-form: methanol:H2O (30:60:8 by vol) and then the acidiclipids were eluted successively in batches with ten columnvolumes of 0.05M, 0.1M and 0.5M ammonium acetate inmethanol. The fractions were desalted using a CIS sep-PackCartridge.

Lipids were chromatographed on aluminium-backed high-performance thin-layer chromatography plates (HPTLC)(silica gel 60, E Merck) either in chloroform: methanol:0.25% KC1 in H2O (5:3:1 by vol), solvent A, or in chloro-form: methanol: H2O (65:25:4 by vol), solvent B.

Chromatographed glycolipids were visualized with 0.5%orcinol in 2 M H2SO4 or with Azur A reagent (Green andRobinson, 1960; Kean, 1968) which is specific for the sulfategroups on carbohydrates.

Lipid antigens were then analysed by immunostaining ofchromatograms with the monoclonal antibody 04 as describedpreviously (Magnani et al. 1982). Hybridoma supernatantcontaining the antibody 04 was diluted 1:4 with buffer A(0.05 M Tris, 0.15 M NaCl pH7.8 containing 1% bovine serumalbumin (BSA) and 0.01 % sodium azide) and the HPTLCplate was incubated in the mixture for 3-5 h at room tempera-ture or overnight at 4°C. After washing in buffer A, the platewas incubated with 2x10*^5 min"1 ml"1 of 125I-labelled goatanti-mouse IgM antibodies (lodogen method). After lh atroom temperature, the plate was washed in cold phosphate-buffered saline (PBS), dried and exposed to Kodak XAR 5X-Ray film. After exposure to X-ray film glycolipids onHPTLC plates were visualized with Orcinol reagent.

Glycolipid antigens recognized by the 04 antibody weredesulfated using 0.05 M HC1 in dry methanol at room tempera-ture for 16 h.

In some experiments, antigens contained in the non-alkali-treated upper phase were alkali treated as described above.After neutralisation, lipids were desalted on Sephadex G50 inH2O. Treated glycolipid antigens were then analysed by

108 R. Mir sky and others

immunostaining of chromatograms with the monoclonal anti-body 04 as described above.

ImmunoblottingThis was carried out as described previously (lessen et al.1984) on samples of newborn and adult sciatic nerve and adultbrain, using 04 monoclonal supernatant diluted 1:10 with 3 %hemoglobin in PBS. The second layer was 125I-labelled rabbitanti-mouse Ig (Amersham International pic).

Results

Appearance of04+ and A007 during the developmentof the rat sciatic nerve and cervical sympathetic trunkTo determine when 04 binding first appears on Schwanncells during the development of the rat sciatic nerve,sciatic nerves were dissected from rats at various agesfrom embryo day 15 to postnatal day 1 and dissociated.Cells were plated on to coverslips and immunolabelledwithin 2 h using 04 antibodies. The results are shown inFig. 1. Briefly, very few (0<0.5%) 04+ cells werepresent at embryo day 15 to 16, whereas over 95 % of allthe cells in the nerve were 04+ on the first postnatal day.To demonstrate that the 04+ cells are restricted to theSchwann cell lineage, cells from embryo day 20 andpostnatal day 1 sciatic nerves were double labelled withS-100 and 04 antibodies. At embryo day 20, 99% ofS-100+ cells were also labelled with 04 antibody and atpostnatal day 1, this figure was 100 % of S-100+ cells.No 04+, S-100" cells were seen at these time pointsshowing that 04+ cells are restricted to the Schwann celllineage.

To test whether another antibody that is highlyspecific for sulfatide gives similar results to thoseobtained with 04 antibody, these experiments wererepeated using rat monoclonal A007-sulfatide antibody.

1 0 0

8 0

6 0

2 0

16 18 20

d a y no.

Fig. 1. Development of 04 antigen on Schwann cells in vivoin rat sciatic nerve. Sciatic nerves of rats from embryonicday 16 to postnatal day 0 were dissociated and labelled withantibodies to 04. The percentage of 04-positive cells at eachtime point is shown on the graph. Results at each timepoint were obtained from a minimum of three experiments,with a total of at least 1000 cells counted.

The results were essentially similar, with few positivecells present at embryo day 16, 35.3% labelled at E17and over 95 % of cells labelled on the first postnatal day,all of which were also labelled with S-100 antibodies.

The appearance of the first 04+ cells, or cells bindingthe A007 sulfatide antibody at embryo day 16, precedesthe appearance of the first galactocerebroside"1"Schwann cells by two days. Several experiments werecarried out to confirm that 04 and A007 binding appearssignificantly before galactocerebroside is detectable. Inone experiment in which cells from embryo day 18 weredouble labelled with 04 and galactocerebroside anti-bodies, 55 % of the cells present were 04+ while nonewere galactocerebroside . In another experiment,93 % of cells from embryo day 20 were 04+ and of the04+ cells only 71% were also galactocerebroside+.Furthermore, at postnatal day 0 all Schwann cells bind04 and A007 sulfatide antibodies (Fig. 2), whereasprevious work has shown that approximately 60% ofSchwann cells are galactocerebroside"1" at this time(Mirsky et al. 1980; Jessen et al. 1985). We also foundthat in the cervical sympathetic trunk 93 % of Schwanncells were 04+ at embryo day 20, and of these only 8 %were galactocerebroside+ in agreement with a previousstudy of galactocerebroside appearance in this nerve(Jessen et al. 1985).

In adult rats Schwann cells of both myelinated andunmyelinated fibres are 04 and A007 positiveThe distribution of 04 and A007 labelling on Schwanncells in adult rats was investigated using teased nervepreparations. The Schwann cells of unmyelinated fibreswere selectively labelled with antibodies to N-CAM(Mirsky et al. 1986) and the myelinated fibres wererecognized morphologically. Both non-myelin-formingand myelin Schwann cells were labelled with 04 anti-body (Fig. 3). These experiments were repeated withA007 sulfatide antibody, which gave similar results withboth myelinated and unmyelinated fibres beinglabelled.

Schwann cells from developing sciatic nerves do notexpress 04 or A007 binding on schedule in vitroTo study the regulation of 04 antigen expression we firstasked whether cells that have not yet become 04+ invivo would become 04+ on schedule in culture.Schwann cells were prepared from embryo day 15sciatic nerves, placed in culture and examined with 04antibodies after 2 days and 5 days. No 04+ cells werefound in these cultures. The same results were obtainedwhen the cells were examined with A007 sulfatideantibodies. This suggests that the triggering of 04 andA007 antigen expression is not intrinsically pro-grammed but depends on extrinsic signals.

Surface expression of 04 and A007 binding bySchwann cells depends on appropriate axonal contactand is regulated by cAMPFurther studies on the regulation of 04 and A007binding were carried out on cells from postnatal nervesusing methods similar to those used previously in

Prenatal Schwann cell development 109

Fig. 2. Double-label immunofluorescence using 04antibodies and S-100 antibodies in a dissociated cell culturefrom newborn rats, 3h after plating. (A) Rhodamine opticsto visualise 04-antigen; (B) phase contrast; (C) fluoresceinoptics to visualise S-100. Note that all S-100-positive cellsare also 04 positive. The three arrows point to fibroblasticcells that are unlabelled with both 04 and S-100 antibodies.Bar: 20 fxm.

Fig. 3. Double-label immunofluorescence using 04antibodies and N-CAM antibodies in a teased preparationof sciatic nerve from a 35 day rat. (A) Rhodamine optics tovisualise 04-binding, (B) phase contrast, (C) fluoresceinoptics, to visualise unmyelinated fibres with N-CAM. Notethat both myelinated and unmyelinated fibres bind 04antibody. Bar: 20fim.

110 R. Mirsky and others

1 0 0

2 4 6 8

d a y s In c u l t u r e

Fig. 4. Disappearance of 04-antigen from Schwann cells indissociated cell cultures of sciatic nerve. Cells were doublelabelled with 04 and S-100 antibodies at different times invitro as described in Materials and methods. Results at eachtime point were obtained from a minimum of threeexperiments with a total of at least 1000 Schwann cellscounted.

studies on the regulation of galactocerebroside andmyelin associated proteins (Mirsky et al. 1980).Schwann cells dissociated from sciatic nerves of 5 dayold rats were placed in culture and 04 labelling moni-tored over the next few days. It was found that theSchwann cells, all of which initially were 04+, slowlylost surface 04 binding so that by day 8 only 0.2±0.2 %of the Schwann cells were still positive (Fig. 4). Whenthese experiments were repeated with A007 sulfatideantibody similar results were obtained, less than 1 % ofS-100+ cells being labelled at 5 days in culture. Theseobservations suggested that the ability of Schwann cellsto bind 04 or A007 antibodies depends on signals fromaxons, or possibly on other factors in the endoneurium.

To show that the level of 04 antigen expression in vivois likely to be regulated by axonal contact, rather thanby other endoneurial factors, the 04 binding was exam-ined on Schwann cells deprived of axonal contact invivo by nerve transection. Schwann cells were removedfrom the distal stump of the sciatic nerve at varioustimes after nerve cut, and 04 labelling monitored byimmunohistochemical methods. It was found that 04binding was substantially down-regulated on the surfaceof Schwann cells in the distal stump, which are deprivedof contact with axons (Fig. 5), although traces of 04labelling were still detectable at high magnificationwhen examined eight weeks after nerve cut. By con-trast, in nerves in which reinnervation had occurredafter a crush lesion, 04 labelling was found associatedwith both myelinated and unmyelinated axons. Rein-nervation was judged to have occurred by return offunction in the leg muscles and by the morphologicalappearance of typical myelinated fibres and unmyelin-ated fibres in teased nerve preparations from thereinnervated distal stump.

In freshly dissociated cultures of dorsal root ganglia

(DRG) from 8 day old rats, 04+ cells also disappearover the first eight days in culture. These culturescontain regenerating neurites which clearly are unableto provide the signal provided by mature axons in thenerve, behaviour also seen in the case of the disappear-ance of galactocerobroside from Schwann cells infreshly dissociated DRG cultures (Mirsky et al. 1980).

Evidence has suggested that cyclic AMP is an import-ant second messenger in Schwann cells, mediating someof the effects of axons on Schwann cell differentiation.We therefore tested whether cAMP analogues andagents such as cholera toxin and forskolin, whichelevate intracellular cAMP, would induce re-expressionof the antigen(s) recognized by the 04 and A007sulfatide antibodies. It was found that these treatmentsinduce both 04 and A007 binding on a substantialproportion of cultured Schwann cells from both post-natal day 5 and embryo day 15. At postnatal day 5,Schwann cells treated for 72 h with cAMP analogues,cholera toxin or forskolin were 88±2% 04+ (ana-logues), 78±1 % 04+ (CTx), and 36±11 % 04+ (forsko-lin) while at 6 days over 99 %, 89 % and 89 % respect-ively were 04+. Corresponding figures for the inductionof A007 sulfatide binding at 72 h were 73±8 %, 36±5 %and 12±6%, respectively. At embryo day 15, thenumber of Schwann cells induced by forskolin was60-70 %04+ and 20-30 %A007+, at 72 h post-treat-ment. The same hierarchy of efficiencies is seen ininduction of galactocerebroside, with cAMP analoguesbeing relatively more potent than either cholera toxinor forskolin (unpublished observations).

04 antibody recognizes sulfatideIt has been reported that the 04 antibody recognizespurified sulfatide in dot blots and on immunolabellingof TLC plates using lipid extracts from the Schwann cellline D6P2T, the two characteristic sulfatide bands arealso recognized (Singh and Pfeiffer, 1985; Bansal andPfeiffer, 1987). Results using polychlonal sulfatide anti-bodies, characterised using different methods, have,however, reported that, in both rat Schwann cells andoligodendrocytes, sulfatide appears on the cell surfaceafter galactocerebroside (Ranscht et al. 1982), whereas04 antigens appear before galactocerebroside (Sommerand Schachner, 1981; Schwab and Caroni, 1988;Wolswijk and Noble, 1989). We therefore decided toreinvestigate this point using extracts from sciaticnerves of newborn and adult rats to establish theidentity of the lipid antigen recognized in the rat PNS.

Lipids extracted from newborn and adult sciaticnerves, and adult rat brain were analysed by immuno-labelling of chromatograms with the monoclonal anti-body 04.

It was clear that 04 antibody bound strongly to aglycolipid with the same chromatographic mobilities aspurified sulfatide and not significantly to any otherglycolipids isolated from these tissues (Fig. 6). In com-mon with many glycolipids sulfatide runs as either oneor two bands in most TLC solvent systems (see Figs 6,7, 8). The bands correspond to differences in theceramide moiety of the glycolipid. By chemical staining

Prenatal Schwann cell development 111

Fig. 5. Double-label immunofluorescence using 04 and S-100 antibodies on cells from normal and transected sciatic nerve.(A-C) Myelin-forming Schwann cell dissociated from 35 day rat sciatic nerve dried on to a microscopic slide; (D-F)Schwann cells dissociated from the distal stump of a transected sciatic nerve 4 weeks after transection, dried on to amicroscope slide. (A,D) Rhodamine optics to visualise 04-antigen; (B,E) phase contrast; (C,F) fluorescein optics to visualiseS-100. Note that 04 binding is easily visible on the single myelin-forming Schwann cell from normal nerve in (A), nucleusarrowed in B. By contrast, it is barely detectable on the three Schwann cells from the distal stump seen in D. The threenuclei of these cells are arrowed in E. S-100 is easily detectable on the Schwann cell from normal nerve (C), and also on theSchwann cells from the distal stump (F). Both sets of pictures were exposed and processed under identical conditions. Bar:20 pm.

with orcinol (Fig. 6B), the molecular species of sulfa-tide corresponding to the lower band appears moreabundant than the more diffuse upper band in the ratnervous tissues tested here, particularly in the newbornsciatic nerve when very little of the upper band isdetectable. 04 monoclonal antibody binds to both

sulfatide species although the affinity for the two formsappears to be slightly different depending on theloading of lipid on the chromatogTam and the solventsystem used. In Fig. 6A the heavier loadings of antigenused in lanes 1, 3 and 4 result in very low, pseudonega-tive staining of the lower band, which is, however

112 R. Mirsky and others

A B

CMH

'CDH

1 2 3 4 5 1 2 3 4 5

Fig. 6. Binding of 04 antibodies in rat nervous tissue.Lipids from 10-20 mg (wet weight) of tissue, present in theupper phase were chromatographed using solvent A. Inpanel A lipid antigens are visualised by immunolabelling ofchromatograms with 04-antibody. In panel B total lipids arevisualized with orcinol reagent. Lane 1: purified sulfatide;lane 2: newborn rat sciatic nerve; lane 3: adult rat sciaticnerve; lane 4: adult rat brain; lane 5: purified neutralglycolipids; CMH ceramide monohexoside (galactosylceramide); CDH: ceramide dihexoside (lactosylceramide).The asterisk indicates the origin of the TLC and two arrowsindicate the positions of the sulfatide bands. Note that thereis 04 labelling in both the sulfatide positions in lanes 1, 3and 4 (Panel A) although saturation of the silica gel by theantigen appears to result in pseudonegative labelling of thelower band in these tracks. The lower sulfatide band iseasily visualised in the less heavily loaded sample ofnewborn sciatic nerve, (track 2) where little labelling of theupper band is present. Labelling at the origin of the TLC inlane 4 is not specific since it is radiolabelled even when 04antibody is omitted during immunolabelling. The extraupper band in Panel A, lanes 3 and 4 is an artefact ofchromatography since it disappears when the lipids aredesalted on a Sephadex G-25 column (see Figs 2 and 3). Bychemical staining (Panel B), the molecular species ofsulfatide corresponding to the lower appears more abundantthan the diffuse upper band in the rat nervous tissue tested(lanes 2-4), particularly in the newborn tissue.

clearly visualised by immunolabelling in Fig. 7C, andFig. 8.

To further characterize this antigen, the glycolipidspurified from different tissues were chromatographed intwo different solvent systems and then either chemicallystained with Azur A, which specifically detects sulfategroups on carbohydrate, or immunolabelled with 04antibody. In both solvent systems (Fig. 7) the glyco-lipids recognized by the 04 antibody have the same

chromatographic mobility as sulfatide when stainedwith Azur A, and the sulfatide is present in newbornsciatic nerve as well as adult nerve and brain. In bothsolvent systems, only one glycolipid is observed sugges-ting that in rat nervous tissues the 04 antibody detectsonly glycolipid migrating in the sulfatide positions.Furthermore, the active component is resistant toalkaline hydrolysis, and sensitive to treatment withHC1, which removes sulfate groups from carbohydrate(Fig. 8). The antigen molecule is present in both upperand lower phases from the Folch partition eluted froman anion exchange column with 0.1 M ammonium acet-ate. These properties all support the positive identifi-cation of the 04 binding antigen as sulfatide. In newbornsciatic nerve, the activity is associated mainly with thelower of the two ceramide forms, whereas in adultnerve and brain there appears to be a relative increasein the proportion of the upper ceramide form.

To test whether the 04 antibody recognises protein-associated determinants, SDS-polyacrylamide gel elec-trophoresis of extracts of newborn and adult nerve, andadult brain were combined with immunoblotting. Nopositive bands were seen suggesting that the antibodydoes not recognize protein related epitopes in additionto sulfatide.

Discussion

04+ and A007* Schwann cells appear early in nervedevelopmentIn the rat sciatic nerve, Schwann cells binding 04 orA007 sulfatide antibodies were first seen on cellsremoved at embryo day 16. At embryo day 18 between30-50% of cells bound the antibodies, and at birthessentially all Schwann cells were 04+ and A007+.Between embryo days 16 and 18, Schwann cells in thenerve surround large groups of axons and the 1:1segregation characteristic of later development of my-elin Schwann cells has not yet occurred. Furthermore,galactocerebroside+ cells, which represent the first cellsdeveloping along the myelin pathway, are first detect-able at embryo day 18, about two days after the firstappearance of 04+ or A007+ cells. The advent of 04 andA007 sulfatide antibody binding therefore precedes thelater developmental differentiation of the cells intoeither the myelin or non-myelin-forming pathways andis not specifically related to myelination. This con-clusion is strengthened by two observations. First,although all Schwann cells bind 04 and A007 sulfatideantibodies at birth, these cells are a heterogenouspopulation. Only about 60% of them are galacto-cerebroside"1" cells developing along the myelin path-way, while the remainder is a mixture of cells, some ofwhich will subsequently be induced to myelinate andothers of which will develop into non-myelin-formingSchwann cells, (Jessen et al. 1985). Second, in adultnerves, both non-myelin-forming and myelin-formingSchwann cells are 04+ and A007 .

Axons regulate 04 and A007 bindingThe ability of Schwann cells to bind 04 or A007

Prenatal Schwann cell development 113

B

1 2 3 4 1 1 1 2 3 4 1

Fig. 7. Occurrence of sulfatide binding in rat nervous tissues. Lipids from 10-20 fig (wet weight) of tissue werechromatographed using solvent A (panels A and B) or solvent B (panels C and D). In panels A and C, lipid antigens arevisualised by immunolabelling of chromatograms with 04-antibody. In panels B and D glycolipids containing sulfate groupsattached to carbohydrate are visualised with Azur A reagent. Lane 1: purified sulfatide; lane 2: newborn sciatic nerve; lane3: adult rat sciatic nerve; lane 4; adult rat brain. In panels A and C, note that the immunolabelling of nerve extract alwayscoincides with the sulfatide positions (arrowed) in both solvent systems, although as in Fig. 6 only the lower band is labelledsignificantly immunolabelled in the newborn nerve extract. Labelling at the origin is again non-specific (see legend to Fig. 6).In panels B and D, the Azur A reagent visualises sulfate predominantly in the lower of the two sulfatide bands in all of thenervous tissue extracts.

antibodies appears to be controlled by axonal contact ina very similar way to that seen in the well-establishedaxonal regulation of galactocerebroside and myelinprotein expression, (Mirsky et al. 1980; Jessen et al.1987/?; Lemke and Chao, 1988; Trapp et al. 1988).Schwann cells from embryo day 15 to 16 sciatic nervewill not acquire the ability to bind 04 or A007 antibodiesin the absence of axons, and 04+ Schwann cells lose theability to bind 04 antibody if removed from axonalcontact either in situ after denervation or in culture.Similar results were obtained with A007 sulfatide anti-body. Low levels of sulfatide synthesis are, however,detectable in long-term cultures of secondary Schwanncells indicating that synthesis of sulfatide is not com-pletely abolished in the absence of axons (Fryxell,1980). This is completely consistent with the presentobservation that at high magnification traces of 04binding were still detectable on denervated Schwanncells in distal stumps of transected nerves. If crushednerves are allowed to regenerate, the Schwann cellsonce more become 04+ indicating that, in regenerationas in development, axonal contact up-regulates levels of04 binding on the Schwann cell surface.

There is evidence that the effect of axon-associatedsignals on the Schwann cell phenotype can in somecircumstances be mimicked by agents that elevate intra-cellular cAMP levels (Baron van Evercooren et al.

1986; Sobue et al. 1986; Lemke and Chao, 1988;Shuman et al. 1988). Forskolin and cAMP analogueswill, for instance, induce re-expression of surface galac-tocerebroside in cultured neonatal Schwann cells thathave lost surface expression of the molecule due toremoval from axonal contact. Our results show thatexpression of the antigen(s) recognized by the 04 orA007 sulfatide antibodies can also be induced by agentsthat mimic or generate high levels of intracellularcAMP. In the case of the neonatal cells, this representsre-expression of antibody binding, previously lost dueto culturing without axons. In the embryo day 15 cells,however, cAMP induced 04 or A007 sulfatide antibodybinding for the first time, since the cells were removedfrom the nerve before 04+ or A007+ cells appear duringdevelopment. In being inducible by cAMP, the anti-gen(s) recognized by the 04 and A007 sulfatide anti-bodies behaves like other axonally induced Schwanncell molecules, including galactocerebroside and themyelin proteins.

Development of the basal laminaOne developmental event to which the appearance of04 and A007 binding might be linked is the acquisitionof a discrete basal lamina by Schwann cells. Theappearance of the Schwann cell basal lamina has notbeen timed carefully in the rat sciatic nerve but the

114 R. Mirsky and others

1 2 3 4 5Fig. 8. Effect of alkali and acidic treatment on 04 binding.Glycolipids from adult sciatic nerves were purified asdescribed in Materials and methods. Lanes 1-5, upper orlower phase antigens eluted from an anion exchangecolumn (pooled 0.05 M and 0.1M fractions). Lane 1: alkali-treated upper phase antigens; lane 2: non-alkali-treatedupper phase antigens; lane 3: acid-treated upper phaseantigens; lane 4: alkali-treated lower phase antigens; lane 5:alkali-treated lower phase antigens subjected to acidtreatment. Treated antigens were chromatographed usingsolvent A. Chromatograms were then immunolabelled with04 antibody as described under Materials and methods.Arrows indicates the position of the purified sulfatide bandsand an asterisk marks the origin of the TLC. Note that the04 binding is unaffected by alkali treatment but destroyedby acid treatment, behaviour consistent with itsidentification as a sulfatide.

available evidence indicates that this occurs at about thetime that all Schwann cells bind 04 and A007 sulfatideantibodies. The antibody binding studied in the presentexperiments is most likely to be essentially due tosulfatide (see below), and sulfatide has been reportedto bind selectively to laminin (Roberts et al. 1985), acomponent of the Schwann cell basal lamina (Corn-brooks et al. 1983; Bunge et al. 1986; Eldridge et al.1989). It seems possible that the appearance of sulfatidein the Schwann cell membrane could be related tostabilization and formation of the basal lamina.

The identity of the 04 and A007 antigen in Schwanncells and its relationship with galactocerebrosideThe 04 antigen expressed by Schwann cells is very likelyto be sulfatide since no other antigen was recognized ineither TLC or SDS-PAGE immunoblotting of neonatalor adult nerves and since another characterized sul-fatide antibody, A007, gave similar results in theimmunofluorescence experiments. 04 antibodies showsome cross-reactivity with seminolipid in immuno-dotblot assays when the lipid is used at high concentrations(Bansal etal. 1989). When embryo day 15 Schwann cells

were examined with anti-seminolipid antibodies(Goujet-Zalc et al. 1986), 55% of the cells wereimmunolabelled, and showed a characteristic labellingpattern with a few fluorescent dots per cell; similarresults were obtained with embryo day 18 cells (data notshown). Neither 04 nor A007 label any Schwann cells atembryo day 15 and at embryo day 18 the majority ofcells are labelled, showing, particularly in the case of04, a densely speckled immunofluorescence pattern.The contribution of seminolipid to 04 or A007 bindingin the present experiments must therefore be minimal,if any. Although the results of labelling with 04 andA007 antibodies are in broad agreement some smalldifferences can be detected between them. Both anti-bodies detect positive cells at embryo day 16, but thenumber of 04+ cells is always slightly greater than thenumber of A007+ cells in parallel experiments. Thelabelling with 04 antibodies is also stronger. Mostprobably the 04 antibody is binding with greater affinitythan the A007 antibody. It is, however, always possiblethat although the major antigen detected in Schwanncells by both antibodies is sulfatide another unidentifiedantigen is being recognized in addition by the 04antibody.

Assuming that the main 04 antigen is sulfatide, it isperhaps surprising that 04 binding is detectable on thecell surface 2 days before galactocerebroside, sincesulfatide is synthesized from galactocerebroside usingthe enzyme galactosyl sulfotransferase. There is con-siderable evidence, however, for the existence of twoseparate pools of galactocerebroside within cells (Ben-jamins et al. 1982; see Bansal and Pfeiffer, 1987 fordiscussion on this point). One of these pools is sulfatedin the Golgi apparatus to form sulfatide which is thentransported via vesicles to the cell surface (Townsend etal. 1984), and the second by-passes the Golgi andreaches the cell surface via a different mechanism. Ourtests have not revealed why a discrepancy exists be-tween studies using monoclonal antibodies recognizingsulfatide, including 04 antibody and A007 monoclonalanti-sulfatide, and those using rabbit antibodies tosulfatide. Monoclonal antibody studies suggest thatsulfatide appears in development in the cell surfacemembrane of Schwann cells or oligodendrocytes beforegalactocerebroside, while rabbit antibody studies indi-cate that it appears later. One possibility is that therabbit antibodies used in previous studies recognizedprimarily the upper of the two sulfatide species visibleon the TLC plates, whereas the 04-antibody recognizesboth the upper and lower species of sulfatide. Theneonatal nerve contains very little of the upper speciesrelative to the lower species. Thus neonatal Schwanncells would react poorly with antibodies recognizingprimarily the upper species of sulfatide whereas cellsfrom nerves of older animals where higher amounts ofthe upper species are present, would be more reactivein immunofluorescence tests. An alternative possibilitycould be that the monoclonal antibodies are of higheraffinity than the polyclonal antibodies and thus wouldbe capable of detecting relatively lower levels of sulfa-tide in the cell membrane in immunofluorescence tests.

Prenatal Schwann cell development 115

Lipid expression in non-myelin-forming and myelinSchwann cellsOther lipid differentiation antigens in addition to sul-fatide, including galactocerebroside and the 08 and 09antigens, are present on mature Schwann cells in bothmyelinated and unmyelinated fibres (Jessen et al. 1985;1987a; Eccleston et al. 1987). This suggests that theplasma membranes of the two adult Schwann cellvariants differ less in lipid composition than in proteincomposition where there is a distinct difference be-tween the two cell types. Thus non-myelin-formingSchwann cells in adult nerves express the cell adhesionmolecules N-CAM and Ll/NgCAM, the cell surfaceproteins 217c(Ran-l), A5E3 and Ran-2, in addition tothe intermediate filament protein GFAP (Jessen andMirsky, 1984; Jessen et al. 1984; Mirsky and Jessen,1984; Mirsky etal. 1985; 1986; Jessen etal. 1990). Thesemolecules are down-regulated in myelin Schwann cellsshortly after they start to express the major myelinglycoprotein Po, myelin basic protein and MAG (Mar-tini and Schachner, 1986; Jessen et al. 1987a; 1990).Therefore, it appears that it is differences in theregulation of protein expression in the myelin Schwanncell pathway rather than differences in lipid expressionthat generate and maintain the two distinct phenotypes(Jessen et al. 1987a; 1990).

Concluding commentsWe show here that 04 and A007 sulfatide antibodieslabel Schwann cells very early in nerve development(two days prior to galactocerebroside appearance) mostprobably due to binding to cell surface sulfatide. Theability of Schwann cells to bind significant levels ofthese antibodies depends on the cells retaining contactwith axons. This indicates that expression of surfacesulfatide in Schwann cells is up-regulated by axon-Schwann cells signals, as previously shown for galacto-cerebroside and the myelin proteins.

Expression of myelin proteins in cultured Schwanncells can be induced by elevation of intracellular cAMPlevels and it has been suggested that cAMP is involvedas a second messenger in the axonal induction ofmyelination. Elevation of cAMP, however, also in-duces Schwann cells to express surface galactocerebro-side and, as indicated in the present work, sulfatide.Neither molecule is specifically associated with themyelin phenotype. Therefore we suggest that if cAMPhas a role in Schwann cell development that role isunlikely to be restricted to myelination, but is probablya more general one extending to the maturation of bothSchwann cell types. The observation that cAMP ana-logues also increase the synthesis of basal laminacomponents in cultured Schwann cells furtherstrengthens this conclusion.

We would like to thank Dr J P Brockes, Dr P A Eccleston,Dr K L Fields, Dr C Goridis, Dr B Ranscht, Dr M Schachner,Dr 1 Sommer and Dr K Stefansson for gifts of antibodies andMs Katayoun Rezvani for help with experiments on embry-onic Schwann cells. This work was supported in part by grantsfrom the Medical Research Council of Great Britain, the

Multiple Sclerosis Society, Action Research for the CrippledChild and Association Francaise contre les Myopathies.

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(Accepted 14 February' 1990)