Development 108, 97-106 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

97

Developmental patterning of the carbohydrate antigen FC10.2 during early

embryogenesis in the chick

WENDY LOVELESS1, RUTH BELLAIRS2, SUSAN J. THORPE1*, MARK PAGE2 t and TEN FEIZI1*1 Section of Glycoconjugate Research, Clinical Research Centre, Watford Road, Harrow, Middlesex, HAI 3UJ, UK2Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E6BT, UK

Present addresses: * Division of Haematology, and t Division of Immunobiology, National Institute for Biological Standards and Control,Blanche Lane, South Mimms, Herts, EN6 3QG, UKt Author for correspondence

Summary

An oligosaccharide antigen (FC10.2), formerly de-scribed only in mammalian cells and secreted glyco-proteins, has been detected and found to display strikingtemporal and spatial patterning in the chick during earlyembryonic development. This antigen is expressed ontype 1 chains, which are isomers of oligosaccharides ofthe poly-A'-acetyllactosamine series (type 2 chains). Ini-munoreactivities before and after neuraminidase treat-ment of serial sections of chick embryos during the first17 stages of development indicate that the FC10.2structure occurs predominantly in the sialylated form(S-FC10.2). The FC10.2 and S-FC10.2 antigens areprominent markers of the primordial germ cells, beingstrongly expressed by these cells from the pre-primitivestreak stage onwards. S-FC10.2 is also a clear marker ofthe pronephric duct from its first appearance. Initiallypresent over the entire apical surface of the ectoderm,antigenicity diminishes in an antero-posterior directionas neurulation proceeds. A unique pattern for a carbo-

hydrate antigen is displayed by cells of the primitivestreak; antigenicity is lost with de-epithelialisation andingression, but is regained in a pericellular distributionon the mesoderm cells that emerge from the primitivestreak. Thereafter, successive changes in expression anddistribution of FC10.2 and S-FC10.2 are features ofmesodermal tissues, particularly during somitogenesis.These antigens are prominent components of the extra-cellular matrix around the notochord and sclerotomecells. They are also prominent posteriorly in the subecto-dermal region, ceasing abruptly at the lateral limits ofthe embryo proper. Although no absolute correlationscan yet be made, several features of the distribution ofthese antigens suggest that they may be integral com-ponents of, or ligands for, cell adhesion molecules.

Key words: carbohydrate antigens, chick embryo,differentiation markers, primordial germ cells.

Introduction

Oligosaccharides are prominent components of cellsurfaces. Marked changes in their structure and distri-bution have been revealed in human and mouse em-bryos with antibodies which recognize specific oligosac-charide sequences (Feizi, 1985). Using antibodiesspecific for linear and branched oligosaccharides of thepoly-A'-acetyllactosamine series (see Table 1) as immu-nohistochemical reagents, it was observed (Thorpe etal. 1988) that the chick embryo resembles the humanand mouse embryos in being rich in oligosaccharides ofthis series. Moreover, striking temporal and spatialpatterning in the expression of these antigens and oftheir sialylated forms was detected in chick embryosinvestigated during the first 17 stages of development.Each antigen showed distinct patterns of expression,

some of which could be correlated with morphogeneticevents.

Two of the main oligosaccharide backbone structuresof glycoproteins, also found on glycolipids, consist ofrepeating disaccharide units of galactose andA'-acetylglucosamine (poly-A'-acetyllactosamine). Dif-ferent linkages of the non-reducing terminal galactoseresidue give rise to the type 1 (galactose linked ySl—3)and the type 2 (galactose linked /Sl-4) oligosaccharidechains (Watkins, 1980). Changes in chain length andbranching patterns of the backbones, and peripheralsubstitutions with various monosaccharides result in awide range of potential cell recognition structures.Mapping of denned backbone structures and theirvariously substituted forms is therefore important forunderstanding biochemical changes and control mech-anisms during embryogenesis.

98 W. Loveless and others

Table 1. Oligosaccharide antigens recognised by sequence-specific antibodies used in the present and previousstudy (Thorpe et al. 1988) of the early chick embryo

Antigen Oligosaccharide sequence Means of detection

I(Ha) Galpl-4GlcNAcpi6Gal/GalNAc

Anti-I(Ha)

Sialosyl-I(Ha) SA-Gaipi-4GlcNAcpl6Gal/GalNAc

New or enhanced anti-I(Ha) immunoreactivityfollowing neuraminidasetreatment

I(Step) ±(Gaipi-4)GlcNAcpi

Galpl-4GlcNAcpi

Galpi-4GlcNAcpl- Anti-I(Step)

Sialosyl-I(Step)

SA

r ±(Gaipi-4)GlcNAcpl

Galpl-4GlcNAcpi-

Galpl-4GlcNAcpl/

New or enhanced anti-I(Step) imnunoreactivityfollowing neuraminidasetreatment

Gaipi-4GlcNAcpl-3Gaipi-4GlcNAcpi-3Gaipi-4GlcNAcpi- Anti-i(Den)

Sialosyl-i SA-Gaipi-4GlcNAcpi-3Galpi-4GlcNAcpl-3Galpl-4GlcNAcPl- New or enhanced anti-i(Den) immunoreactivityfollowing neuraminidasetreatment.

FC10.2 Gaipi-3GlcNAcPl-3Galpl-4Glc/GlcNAcpl- Antibody FC10.2

Sialosyl-FC10.2(S-FC10.2)

SA-Gaipi-3GlcNAcpl-3Galpi-4Glc/GlcNACpi- New or enhanced anti-body FC10.2 immuno-reactivity followingneuraminidasetreatment.

B-related Galal-3Gaip-I

±(Fucal-2)

Antibody NB10.3B4

Abbreviations: Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc,N-acetylglucosamine; SA, sialic acid. ± indicates monosaccharides which may be present but which do notinfluence immunoreactivity.

In the present study the expression and distributionof the type 1 backbone have been investigated in thechick embryo during the first 17 stages of development.This backbone structure (see Table 1) is recognized by a

hybridoma antibody FC10.2, which was originallyraised against a human embryonal carcinoma line LICRLON HT 39/7 (Cotte et al. 1982). The epitope for thisantibody is a tetrasaccharide, detectable as an overtly

expressed antigen in human embryonic endoderm (Wil-liams et al. 1982), but masked (Gooi et al. 1983) invarious adult epithelial cells and secreted glycoproteinsby the addition of the blood group monosaccharides. Inthe early mouse embryo, FC10.2 antigen has beendetected only in the embryonic endoderm and the yolksac (Pennington et al. 1985). We now report that theFC10.2 antigen and its sialylated form, designated hereas S-FC10.2, also occur in the chick embryo, and aredetectable from the unincubated stage onwards withdistinct patterns of distribution, including an associ-ation with certain migratory cells.

Materials and methods

Chick embryosHens' eggs were either unincubated or were incubated forperiods up to 72h (stages 1-17 of Hamburger and Hamilton,1951). Embryos were dissected from the yolk and membranesin Pannett and Compton's saline (Pannett and Compton,1924), fixed in buffered formal-saline (Drury and Wallington,1967), dehydrated in ethanol concentrations, embedded inparaffin wax, serially sectioned at 4j«n and mounted onethanol-washed glass slides.

Monoclonal antibodiesThe mouse hybridoma antibody FC10.2 of IgM class (a giftfrom Dr R. A. J. Mcllhinney, MRC AnatomicalNeuropharmacology Unit, Oxford, UK) raised against theformalin-fixed human undifferentiated teratocarcinoma cellline LICR-LON 39/7 (Cotte et al. 1982), recognizes theoligosaccharide sequence Gal/31-3GlcNAc£l-3Gal/Sl-4Glc/GlcNAc (Gooi et al. 1983), which occurs as the type 1backbone of the human blood group A, B and H antigens(Watkins, 1980). A mouse hybridoma antibody NB10.3B4, ofIgM class (a gift from Dr E. D. Lennox, Celltech, Slough,UK), was used as a control antibody. This antibody reactsequally well with untreated and de-fucosylated group Bovarian cyst substances (H.C. Gooi, unpublished obser-vation), and recognizes the blood group B-related sequenceshown in Table 1. Both FC10.2 and NB10.3B4 were used asundiluted culture supernatants.

JmmunohistochemistryParaffin wax-embedded sections were stained without treat-ment or after treatment with 0.2 units ml"1 sialidase fromVibrio cholerae as described previously (Thorpe et al. 1988),except that the diluent for the enzyme was 0.15M-acetatebuffer, pH5.5, containing 1 mM-calcium chloride. Boundantibodies were detected either by indirect immunofiuor-escence (Thorpe el al. 1988), or by using goat anti-mouse IgMbiotin-avidin-horseradish peroxidase Vecta ABC kit (VectorLaboratories, Burlingame, CA 94010, USA), following themanufacturer's recommendations. Peroxidase-stained sec-tions were counterstained with 0.02% Light Green, mountedin DPX (Nustain, Nottingham, UK), and photographedunder bright-field using Kodak Ektachrome 64 film. Withantibody FC10.2, an increase in immunoreactivity or theappearance of new areas of reactivity following treatment ofthe sections with sialidase was taken to indicate the presenceof the sialylated structure, S-FC10.2.

Oligosaccharide patterning in embryogenesis 99

Results

The carbohydrate antigen FC10.2 and its sialylatedform S-FC10.2 were detected in the chick embryo at allstages examined, from the unincubated egg (stage 1)until stage 17 (30 pairs of somites). In the unincubatedembryo (Fig. 1A) these antigens were detected at theapical side of the ectoderm and in the primordial germcells. From the primitive streak stage (Fig. IB)onwards, several striking patterns of cell surface andextracellular matrix reactivity were observed. S-FC10.2was the predominant form; the non-sialylated FC10.2,where expressed, was usually weaker or not apparentuntil a later stage (Table 2).

Persistent antigenicity in primordial germ cellsPrimordial germ cells, identified by their size (diam-eters up to 20/m\) and location, showed strong intra-cellular and pericellular reactivity for FC10.2 andS-FC10.2 antigens. They were conspicuous either singlyor in groups beneath the ectoderm at stage 1 (Fig. 2A),in the germinal crescent from stage 5-8 (Fig. 2B andC), in the blood vessels from stage 10-14 (not shown)and in the splanchnic mesoderm associated with the gutfrom stage 15-17 (Fig. 2D). Cells with similar charac-teristics were detected in the ectoderm of the pre-primitive streak stages (Fig. 2A).

Transient loss of expression in cells migrating throughthe primitive streakCells which had left the ectoderm and ingressed to formthe primitive streak no longer expressed FC10.2 orS-FC10.2. However, on those cells that had migratedfrom the primitive streak to form paraxial mesoderm,S-FC10.2 reappeared in a pericellular distribution(Fig. 3A). This pericellular staining was also a charac-teristic of the later derivatives of the primitive streak,i.e. the mesoderm cells of the tail bud, from stage 16(Fig. 3B).

Changes of expression in ectodermal tissuesCells destined to become neural plate were antigen-negative from stage 5 (Fig. IB). Cells derived fromthese, forming the neural plate, and later the neuraltube, remained virtually antigen-negative (Figs 4 and5A) giving rise to a progressive antero-posterior exten-sion of the antigen-negative state.

By contrast, in cells destined to become non-neuralectoderm there was a persistence of S-FC10.2 anti-genicity until stage 8. Thereafter, there was a gradualloss (Figs 1C-E, 4 and 5A) in an antero-posteriordirection until stage 17 when small patches of anti-genicity remained in the tail bud. Meanwhile, restrictedregions of S-FC10.2 reactivity were detectable at theventral surface of the head ectoderm and over the lensplacodes (Fig. ID and E; Table 2).

Changes during differentiation of axial and paraxialmesodermCells of the notochord showed a strong pericellularreactivity for S-FC10.2. At every stage the reactivity

100 W. Loveless and others

A

-ao

B

hP

ao

Stage 1

np ao

ps

- - / / - - h

Stage 14

was more intense in the anterior regions of the noto-chord. From stage 14 there was additional reactivity inthe surrounding notochordal sheath especially at theanterior end (Fig. 5A, B and C).

Cells of the segmental plate mesoderm also showedpericellular staining. There was an antero-posteriorgradient of antigenicity with the greatest intensity at theposterior end (Figs 2B and 6).

In the newly segmented somites, where the cells hadbecome epithelialised, there was a redistribution of theantigenicity to the basal and luminal surfaces (Fig. 5D).In the more mature somites, further changes were seenin the antigen distribution: in the sclerotome region,derived from the ventro-medial aspect of each somite,S-FC10.2 had an extracellular distribution (Fig. 5E)

Stage 17

Stage 10

Fig. 1. Diagram to illustrate theexpression of S-FC10.2 antigen(shaded) at the apical surface of theectoderm of chick embryos atstages 1 and 5, and the non-neuralectoderm at stages 10, 14 and 17.For orientation purposes, thedrawings include several non-ectodermal tissues, indicated bydotted leader lines. In B-E theperipheral limits of the area opacaor yolk sac are not included.Abbreviations: ao, area opaca;ap, area pellucida; fb, fore brain;h, heart; hp, head process; 1, lens;lp, lens placode; np, neural plate;nt, neural tube; ps, primitivestreak; s, somites; sp, segmentalplate; tb, tail bud.

which merged into that of the notochordal sheath(Fig. 6B). At the dorsal side of the somites, S-FC10.2was found at the luminal (apical) border of the derma-myotome (Fig. 6B).

At stage 13 when the pronephric duct could berecognised, its cells stained strongly for S-FC10.2(Fig. 5A).

Patterning in relation to body folds and the futureamnionA conspicuous reactivity and patterning of S-FC10.2was apparent at stage 14 in the extracellular matrixunderlying the embryonic ectoderm. This extendedover the posterior somites and the medial part of the

\

e pgc

"t

pgc

m

B

gc

m Hn

SpA77

CC

spm

Fig. 2. Expression of S-FC10.2 antigen in primordial germ cells detected by immunoperoxidase staining. (A) Stage 1,section of embryo showing immunoreactivity of a germ cell beneath the ectoderm of the area pellucida. Note a cell withsimilar reactivity within the ectodermal layer (arrowed; X636). (B) Stage 7, sagittal section showing an area (boxed)containing primordial germ cells in the germinal crescent anterior to the head fold (X78). (C) Enlargement of germinalcrescent (boxed region in panel B; X636). (D) Stage 17, parasagittal section showing primordial germ cells clustered in thesplanchnic mesoderm (x778). Abbreviations: cc, coelomic cavity; e, ectoderm; en, endoderm; gc, germinal crescent;Hn, Hensen's node;m, mesoderm; pgc, primordial germ cells; spm, splanchnic mesoderm.

B

Fig. 5. Patterns of expression of S-FC10.2 in notochord (A,B,C), somites (D,E) and pronephric duct (A) detected byimmunoperoxidase staining. (A) Transverse section of mid-trunk of a stage 15 embryo. In the notochord, pericellularstaining only is visible (xl94). (B) Transverse section of the notochord at the anterior end of a stage 14 embryo showingintercellular and perinotochordal staining (X566). (C) Oblique section of notochord from posterior end of the same embryoshows intercellular staining only (x566). (D) Longitudinal section of an epithelial somite from a stage 14 embryo (x409).(E) Longitudinal section of a mature somite from the same embryo (x409). Abbreviations: bs, basal surface; d, dermatome;e, ectoderm; lu, luminal surface; n, notochord; nt, neural tube; p, pronephric duct;s, somite; si, sclerotome.

Oligosaccharide patterning in embryogenesis 101

Table

Tissues

2. Distribution ofFC10.2 and S-FC10.2antigens

Regions

in chick embryos during the first 17 stagesStages

FC10.2 S-FC10.2

PRIMORDIAL GERM CELLS

EPITHELIA"Ectodermal

Endodermal

Mesodermal

MESODERMMesenchymal

EXTRACELLULAR MATERIALS

Area pellucida, germinal crescent, blood vessels,splanchnic mesoderm

"Antigen expressed at apical surface."Parentheses indicate weak expression.cN=not detected.d Antero-posterior gradient, most intense posteriorly.8 Antero-posterior gradient, most intense anteriorly.fOver epithelial somites and medial lateral plate.

1-17 1-17

AllNeuralNon-neural (anterior)

(posterior)Head ectoderm (patchy)Placodes

Pharynx (including cytoplasm)Gut roofBeneath splanchnic mesoderm (trunk)HindgutExtraembryonic (patchy)Somatic lateral plate:-

amnioticarea opaca

Splanchnic lateral plateEpithelial somitesDermatomePronephric duct

Lateral to primitive streakSegmental plated

Tail bud

Between notochord cells"Around notochorde

Between sclerotome cellsBeneath dermatomeSubectodermal'Subectodermal in tail regionAmniotic fluid

(1-4)N*

(1-8)(1-17)16-17

N

N(10-17)

NNN

14-171717N

(17)N

N14-1716-17

14-17NN17

16-1717

14-17

1-4N

1-81-17

12-1716-17

14-178-178-1717

4-17

12-1713-1712-1712-1612-1713-17

4-147-17

16-17

8-1714-1714-1714-1714-17

1714-17

lateral plate, ceasing abruptly at the lateral body folds(Fig. 4A). This subectodermal reactivity extended intothe tail bud (Fig. 3B).

In the extraembryonic lateral plate mesoderm, therewas strong FC10.2 and S-FC10.2 reactivity in theamniotic folds (Fig. 4A), which was diminished in theamnion proper. This coincided with the acquisition ofreactivity in the amniotic ectoderm (Fig. 4B). Reac-tivity was also detected as amorphous material in theamniotic cavity (not shown).

Distribution in the endodermVarious regions of the embryonic endoderm showedS-FC10.2 reactivity from stage 8, whilst the extra-embryonic endoderm showed persistent reactivity fromstage 4 onwards (Table 2).

Discussion

This study provides, to the best of our knowledge, thefirst evidence for the presence of the type 1 oligosac-charide backbone structure (recognised by FC10.2antibody) in the chick embryo. Evidence is presentedthat this structure occurs predominantly in the sialyl-ated form with a distinct patterning of distribution ateach stage. There are patterns associated with themigration of certain cells, while others are related to thefolding of epithelial sheets. Some of these patternsresemble those observed for certain of the type 2oligosaccharide structures (Thorpe et al. 1988), whileothers are unique to the type 1 structure.

From the present and earlier studies (Thorpe et al.1988) we deduce that the primordial germ cells are richin both type 1 (FC10.2) and type 2 [anti-i (Den) andanti-I (Step)] antigens. The intense cytoplasmic and

102 W. Loveless and others

Fig. 3. Pattern of expression of S-FC10.2 in cells associated with the primitive streak at stage 8 (panel A) and with the tailbud at stage 16 (panel B) detected by immunofluorescence. In A (x533) immunoreactivity is strong at the apical surface ofthe ectoderm, lacking in the primitive streak, and strong again around the paraxial mesoderm cells. In B (xl57) there isintense peri- and extracellular immunoreactivity in the mesoderm and sub- ectodermal regions. A' and B' are phase-contrastmicrographs of panels A and B, respectively. Abbreviations: e, ectoderm; m, mesoderm; ps, primitive streak.

surface expression of FC10.2 antigen by these cellsallows their detection at the pre-primitive streak stage.Such a distinction could not be made with anti-i (Den)or anti-I (Step), since these antibodies were found toreact with the cytoplasm of all cells at this stage. ThusFC10.2 antigen is a clear marker of the primordial germcells at all stages studied. Another monoclonal anti-body, EMA-1, has been reported to identify these cellsat the pre-primitive streak stage (Urven et al. 1988).The antigenic determinant for this antibody is thoughtto consist of fucosylated type 2 chains (Hahnel andEddy, 1986), and so may be related to the mouseembryonic antigen SSEA-1 (Gooi et al. 1981), which isstrongly expressed on mouse primordial germ cells(Wylie et al. 1986; T. Taketo and T. Feizi, unpublishedobservations). FC10.2 and EMA-1 enable the avianprimordial germ cells to be identified at an earlier stagethan by traditional markers, e.g., staining for glycogen(Clawson and Domm, 1963; Meyer, 1964), and withricin (Didier et al. 1981). Moreover, both FC10.2 andEMA-1 are expressed by scattered cells in the ectodermat the pre-primitive streak stage, which is consistentwith the view (Ginsburg and Eyal-Giladi, 1987) thatprimordial germ cells originate from the ectoderm.

The striking loss of S-FC10.2 reactivity at the apicalside of the ectoderm associated with the formation of

the neural plate closely parallels the loss reportedearlier (Thorpe et al. 1988) of the long chain type 2antigens i (Den) and I (Step), and contrasts with thegain of the short branch antigen I (Ma). These obser-vations, together with the changes in lectin binding(Currie et al. 1984; Takahashi and Howes, 1986; Taka-hashi, 1988), highlight the pronounced glycosylationchanges that accompany neurulation.

The expression of S-FC10.2 in the subectodermalspace resembles that of i (Den) and I (Ma) in that it islocated posteriorly in the embryo and does not extendlaterally beyond the future lateral boundary of theembryo proper, i.e. the medial part of the lateral plate.However, the distributions are not identical; forexample, at stage 14 (see Fig. 7), only S-FC10.2 extendsfurther posteriorly, only i (Den) is associated with theanterior trunk neural crest, and only I (Ma) lies over themost anterior somites.

Pericellular immunoreactivity within the notochord,a feature of the sialylated type 1 antigen, has not beendetected for the type 2 antigens. In the perinotochordalregion both the type 1 (S-FC10.2) and the short-chainbranched type 2 antigen [sialosyl-I (Ma)] are expressed,although their distributions are strikingly different.Thus S-FC10.2 occurs principally in the notochordalsheath, extending to a lesser extent towards the sclero-

Oligosaccharide patterning in embryogenesis 103

I A

B'

Fig. 4. (A) Immunofluorescence of a transverse section through the anterior trunk region of a stage 16 embryo (xlO6).There is strong expression of S-FC10.2 in the sub-ectodermal extracellular matrix, over the somites and the medial part ofthe lateral plate, and between the cells of the somites. Further laterally, the somatic lateral plate mesoderm of the amnioticfold is strongly immunoreactive. The notochord staining is unusually weak in this embryo. (B) Immunofluorescence of asection through the amnion of a stage 17 embryo (x488) showing S-FC10.2 reactivity of the ectoderm. B' is thecorresponding phase-contrast micrograph. Abbreviations: e, ectoderm; lp, lateral plate; n, notochord; nt, neural tube;s, somite; sm, somatic mesoderm of the amniotic fold (A) and of the amnion (B).

tome cells. By contrast, sialosyl-I (Ma) is found in asharply defined region which corresponds to the futurecentra of the vertebrae (Thorpe et al. 1988).

Several features of the type 1 patterning are not seenwith the type 2 antigens. One of the most striking is theS-FC10.2 antigen patterning in relation to de-epitheli-alisation. Thus as the ectodermal cells enter the primi-tive streak they no longer express S-FC10.2, but as theyemerge as mesenchyme cells to form the embryonicmesoderm this antigen is re-expressed. Three ad-ditional differences are observed. First, there is anantero-posterior gradient of FC10.2 and S-FC10.2 inthe paraxial mesoderm with the most intense expressionposteriorly; second, S-FC10.2 is a marker for thepronephric duct from its earliest appearance, and third,in the amniotic folds and the amnion, the type 1antigens are expressed in the mesodermal and ectoder-mal layers, whereas the type 2 antigens are onlydetected in the ectoderm.

Collectively these observations point to a remarkablecontrol of glycosylation during development and raiseimportant questions as to the roles of the individualstructures. If, as has been suggested (Feizi, 1981), eachconstitutes a recognition structure on glycoproteins andglycolipids, it would be predicted that for each therewould exist a complementary carbohydrate-bindingprotein. We suggest that possible candidates among

these are adhesion molecules, of which several havebeen identified. The spatio-temporal patternings of theoligosaccharide antigens have features in common withthose described for adhesion molecules which occur oncell surfaces and in extracellular matrices, and whichappear to have major roles in mediating cell-cell andcell-substrate interactions fundamental to morphogen-esis (Takeichi, 1988; Edelman, 1989). It has beensuggested that these adhesive events are mediated byhomophilic (like-like) and heterophilic interactionsbetween the adhesion molecules, but the precise mol-ecular bases of these interactions are as yet unknown.The results presented here and earlier (Thorpe et al.1988) strengthen the idea that the key structures recog-nised by adhesion molecules may include specific oligo-saccharides. Adhesion molecules are typically glyco-proteins, several of which express carbohydrateantigens (Schachner, 1989). Prominent among thesestructures is HNK-1, a glucuronic acid-containing struc-ture (Chou etal. 1986) based on the type 2 oligosacchar-ide backbone, which has been implicated in cell-celladhesion (Keilhauer et al. 1985), although this stillneeds to be established conclusively. Polysialosyl carbo-hydrate chains which are abundant on the embryonicbut not the adult form of the neural cell adhesionmolecule (Finne, 1985) are thought to modulate thetightness of cell-cell adhesion (Rutishauser et al. 1988).

104 W. Loveless and others

Aecm

Future tb

bs

Fig. 6. Diagram of a parasagittal section through the trunk and tail region of a stage 14 embryo. In the segmental plate theexpression of S-FC10.2 antigen is greatest at the posterior end. In the epithelial somites (shown also in transverse section inC), the immunoreactivity is confined to the basal (outer) surface and the lumen. In the mature somites (located anteriorlyand also shown in transverse section in B), S-FC10.2 reactivity occurs at the apical side of the dermamyotome and betweenthe sclerotome cells, extending as far as the perinotochordal region (see also Fig. 5). In the posterior trunk the apical surfaceof the ectoderm and the subectodermal extracellular material shows reactivity. Abbreviations: bs, basal surface;d, dermatome; e, ectoderm; ecm, extracellular matrix; en, endoderm; lu, lumen; m, myotome; n, notochord;s, somite;si, sclerotome; sp, segmental plate; tb, tail bud.

Den

Neuralcrest

Slalosyl-I Ma

Lateral plateProximal

Distal Head

Heart

Neural tube

Somites

Segmentalplate

Tail bud

Fig. 7. Schematic representation of the subectodermal extracellular space of the chick embryo at stage 14. Anteroposteriorand mediolateral patterns of distribution are shown for the sialylated, linear type 1 backbone structure (sialosyl-FC10.2)observed in the present study, and compared with those for the linear (i Den) and the short-branched (I Ma and sialosyl-IMa) type 2 structures reported earlier (Thorpe et al. 1988).

Oligosaccharide patterning in embryogenesis 105

Here also the precise molecular mechanisms have notbeen elucidated, although recognition by an endogen-ous carbohydrate-binding protein (lectin) cannot beruled out. A growing number of lectins are beingidentified in animal tissues (Barondes, 1988), and inaddition structural features characteristic of lectins(Drickamer, 1988) have been found on a variety ofproteins that may mediate cellular adhesion (Bevil-acqua etal. 1989; Johnston etal. 1989; Lasky etal. 1989;Siegelman et al. 1989). With such developments,together with novel approaches to determine the speci-ficities of mammalian carbohydrate-binding proteins(Childs et al. 1989; Loveless et al. 1989; Mizuochi et al.1989), there are exciting possibilities for elucidating theroles of oligosaccharides in adhesive interactions.

This work was supported by grants from Action Research,the Arthritis and Rheumatism Council, the British HeartFoundation and the Cancer Research Campaign. The authorswould like to thank Dr R. A. J. Mcllhinney for antibodyFC10.2, Mr N. Webb for art work, Mrs R. Cleevely fortechnical assistance, and Mrs M. Moriarty for typing themanuscript.

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