Expression of an endogenous galactose-binding lectin in the early chick

embryo

SARA E. ZALIK, LAWRENCE VV. THOMSON and IRENE M. LEDSHAM

Department of'Analogy, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Summary

The gastrulating chick blastoderm contains lectinactivity specific for /3-D-galactoside groups. Thegalactose-binding lectin isolated by affinitychromatography on p-aminophenyl-/3-D-lactosideseparates into two bands when studied by sodiumdodecyl sulphate-polyacrylamide gel electro-phoresis. One of these Ln has a relative molecularmass of 70(±2)xl03 while the other L̂ is a poly-peptide that migrates with the dye front in 10%gels. We have prepared an antiserum against thislectin preparation and have affinity-purified anti-bodies against Li. When embryos at stages 3-7were examined by immunofluorescence using the

affinity-purified antibodies, lectin was expressedin cells at the lowest portions of the primitivestreak as well as in cells migrating laterally fromthis region to form the endoderm. Lectin was alsoexpressed by the cells of the extra-embryonicendoderm and the primordial germ cells of theproximal area opaca. In transfers of gradient gelsstained with affinity-purified antibodies againstL], this lectin had an approximate molecularweight of 6-5 xlO3. Our results indicate that thislectin is expressed in areas that are undergoingcell spreading.

Key words: galactose, lectin, embryo, blastoderm.

Introduction

Carbohydrate-binding proteins or lectins are widelydistributed in animal organisms (Lis & Sharon, 1986).Changes in lectin activity occur during embryogenesis(Harris & Zalik, 1985) as well as in the development oforgan systems (Barondes, 1984; Zalik & Milos, 1986).In the early chick embryo a galactose-specific lectin isalready present in extracts of preincubated blastoderms(Cook el al. 1979); this lectin has been isolated fromprimitive streak blastoderms (Zalik et al. 1983). Whenexamined by sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS-PAGE) using 10% gels, thislectin separates into two bands. One of these (lectin I)is a polypeptide that migrates with the dye front. Thesecond lectin (lectin II) has a relative molecular mass of70(±2)Xl03, and appears sometimes as a doublet(Zalik et al. 1983).

Cells obtained by mechanical dissociation from dif-ferent areas of the primitive streak embryos havegalactose-bearing receptors at their surfaces, as shownby the agglutinability of these cells by plant lectins(Phillips & Zalik, 1982). From the area opaca of these

Journal of Cell Science 88, 483-493 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

embryos we have isolated pure populations of endodcr-mal cells. These cells are the progenitors of the extra-embryonic epithelium of the yolk sac and in this paperthey will be referred to as extra-embryonic endodermcells (EEC). In vitro, EEC form aggregates thatsubsequently undergo cavitation (Milos et al. 1979).The endogenous lectin decreases the adhesion of sus-pensions of EEC (Milos & Zalik, 1982, 1983). Alsoduring the cellular reorganization that occurs duringaggregate cavitation, lectin is released into the vesicularfluid contents of EEC aggregates (Milos & Zalik, 1986).In this paper we describe one of the antisera that wehave obtained against preparations of the endogenousblastoderm lectin. From this antiserum we have affin-ity-purified antibodies specific for lectin I (L|), andhave used these to study the cellular localization of Liduring gastrulation and early neurulation. Our resultsindicate that lectin is expressed in cellular populationsthat are undergoing cell migration and cell spreading.Using affinity-purified antibodies combined with im-munoblot transfers of gradient gels, an estimate of themolecular weight of Lj has been obtained.

483

Materials and methods

Lectin extraction and purificationCrude lectin extracts were obtained from 23-h incubated eggs(primitive streak blastoderms stages 4-5; Hamburger &Hamilton, 1951), as reported (Zalik et al. 1983). Essentiallythis procedure consists of homogenization of the blastodermsin a lectin extraction solution MEPBS (0-15 M-NaCl, 0-005 M-NaHPO4-KH2PO4, pH7-2, 0-004M-mercaptoethanol) con-taining 0-3M-lactose and 0-25 mM-phenylmethylsulphonylfluoride (PMSF; Sigma) using a TenBroek glass homogen-izer. Following centrifugation (100 000 g, 1 h) extracts weredialysed exhaustively against MEPBS and concentrated. Theaffinity-purified lectin preparations used for immunoblotanalysis were isolated using Affi-Gel 10 (BioRad) coupled,according to the manufacturer's instructions, to p-amino-phenyl-^-D-lactoside (APL) (Vega-Fox Biochemicals). Thelectin preparations used for preparation of the antisera wereobtained from extracts from approximately 3000 blastodermsand purified by affinity chromatography on APL-Sepharoseas reported (Zalik et al. 1983). Lectin activity was assessedusing stabilized trypsinized rabbit erythrocytes (Zalik et al.1983). Protein content was determined according to Bradford(1976) using gamma globulin as a standard.

Preparation of antiserumThe procedure used by Novvak et al. (1977) for the prep-aration of antibodies against the chick pectoral muscle lectinwas used with some modifications-. Nine-month-old rabbitswere injected intradermally at multiple sites in the back. Atotal of 131/ig of purified lectin in Freund's completeadjuvant was given in three approximately equal injections at2-week intervals over a 6-week period. Two weeks later31-5 /.tg of lectin in saline was injected intravenously. Sincethe serum collected 2 weeks after this injection gave no clearprecipitin reaction with crude lectin extracts, additionalintravenous injections of purified lectin were administered. Asecond injection (47 fig) was given 2 weeks after the firstintravenous injection and the third injection (37 fig) wasgiven three and a half months later. When serum collected 10days after the last injection was tested in double gel diffusionagainst crude lectin extracts, it gave rise to two bands, whileno bands were present in control serum (Fig. 1A). Agardiffusion was performed in 35 mm Petri dishes using 0-3%agarose (BioRad) in borate-buffered saline according toGarvey et al. (1977), except that 0-3M-lactose was incorpor-ated into the borate-buffered saline prior to addition andsolubilization of the agarose. Antibody concentration in theantiserum and in the affinity-purified antibodies was assessedby radial immunodiffusion (Mancini et al. 1965). For thispurpose goat anti-rabbit immunoglobulin (Miles) was incor-porated into the agar. Standards consisted of rabbit immuno-globulin (Miles). The precipitin ring was enhanced usingtannic acid (Simmons, 1971) and the area encompassed bythe ring halo was measured with a measuring microscope(Gaertner). The immunoglobulin concentration was calcu-lated by regression analysis (Fig. 1B,C).

Immunoblot analysisCrude lectin extracts we separated by SDS-PAGE using10% or 10% to 17-5% gradient gels, the latter were

stabilized with a 1 % to 10 % sucrose gradient. A 3 % stackinggel was used in both gel types. Crude lectin extracts, purifiedlectin samples or protein standards (Sigma, B.R.L.) wereheated to boiling for 5 min in sample buffer (0-05M-Tris-HC1, pH6-8, 2% SDS, 7% glycerol 4-3% /3-mercaptoetha-nol, 5 M-urea). The electrophoresis buffer (25 mM-Tris- HC1,192mM-glycine, 0-1% SDS) was prepared according toLaemmli (1970). Following electrophoresis gels were trans-ferred to nitrocellulose membranes (BioRad 0-22f(m poresize) according to Towbin et al. (1979), using 25 mM-Tris • HC1, 192 mM-glycine with or without 20 % methanol ona Hoefer Transfer or a Bio-Rad Trans-Blot apparatus. Gels tobe transferred without methanol were allowed to swell in therespective transfer buffer for 20-30 min before transfer.Transfer of L| in the presence of methanol was nearcompletion following 2h at 0-3 A. To permit representativetransfers of the higher molecular weight proteins, gels weretransferred in the absence of methanol for 3h at 1-5-2-0 A.Coomassie Brilliant Blue R-250 (BioRad) staining of verticalgel slices taken before and after transfer, and Amido Blackstaining of vertical slices of nitrocellulose transfers were usedto monitor the efficiency of the transfer. The Amido Blackstain consisted of a 5-min stain in 0-1 % (w/v) Amido Blackin 5 % methanol and 10% acetic acid followed by a waterdestain. Proteins and standards in the transfers were localizedby staining representative vertical slices with Amido Black.

To determine the specificity of the antisera and the affinity-purified antibodies, nitrocellulose strips were blocked with5 % bovine serum albumin (BSA; 98-99% albumin, Sigma)in Tris-buffered saline (TBS) containing 0-05 M-Tris-HC1,0-15M-NaCl, pH7-4, for 2h. All probing steps were carriedout at room temperature. The blots were then incubated withrabbit antiserum diluted 1:400 in TBS containing 3 % BSA,for 14 h followed by four washes, of 20 min each, with TBScontaining 0-05 % Tween 20 (BioRad). Blots were probed for2h with goat anti-rabbit immunoglobulin coupled to horse-radish peroxidase (GAR-HRP) diluted 1:3000 (BioRad) andwashed in TBS as outlined above. Peroxidase activity wasdetected with 4-chloro-l-naphthol substrate (BioRad), 30 mgdissolved in 10 ml methanol and diluted to 50 ml with TBSand made to 0-01% H2O2 (Hawkes et at. 1982). Positivebands gave a blue colour within 1-10 min.

Affinity purificationAntibodies specific for lectin I were purified according to

Olmsted (1981) and Talian et al. (1983). Crude lectinfractions were separated on 10% SDS-PAGE slab gels(Laemmli, 1970) as described above using a comb containingtwo end reference wells interspaced by a wide sample well.Gels were transferred to 0-22fim nitrocellulose and Li wasinitially localized on a representative vertical nitrocellulosestrip using the immunoperoxidase staining procedure out-lined above. In subsequent experiments L| was localized bystaining representative nitrocellulose strips from slab trans-fers with 0-1 % Amido Black. For affinity purification of theantibodies 0-5 cm wide horizontal strips of nitrocellulosecontaining localized L] were cut out, blocked with 5 % BSAin TBS (TBS-BSA) for 2h, incubated with the crudeantiserum diluted 1:400 in TBS-BSA for 14 h at roomtemperature and washed four times as outlined for theimmunoblotting staining. Antibodies were eluted by washing

484 5. E. Zalik et al.

the nitrocellulose strip with 200 mM-glycine • HC1, pH2-8,for 3-4 min with the aid of a pipette. The strips were thenwashed with Dulbecco's phosphate buffered saline (170mM-NaCl, 3mM-KCl, 10mM-Na2HPO4, 2mM-KH2PO4), with aratio of 3:1 (v/v) saline/glycine• HC1. The pooled glycine-saline mixture was brought to pH 7'0 with 1 M-NaOH withinan overall extraction time of 5 min. The extraction wasrepeated three times and the pooled extracts were concen-trated over a ym 10 membrane (Amicon).

A blot-dot procedure (Hawkes el al. 1982) was used tomonitor antibody activity following the various elution pro-cedures. Samples of 1-5/il of the antibody extract or rabbitserum (positive control) were spotted on nitrocellulose sheetsmarked in square grid patterns. The nitrocellulose was thenblocked with BSA and probed with GAR-HRP as describedabove.

Immunofluorescence

Blastoderms at stages 3-7 (Hamburger & Hamilton, 1951)were fixed at room temperature, either for 2h in freshlyprepared 3-7% paraformaldehyde in Pannett & Compton's(1924) saline (PCS), p H 7 4 , or for 1 h in absolute ethanol.The embryos were dehydrated with and embedded in poly-ethylene glvcol (PEG) by a modification of the procedure ofDrews (1975). Embryos were rinsed briefly in PCS followedby distilled water and dehydrated sequentially in 50%, 75 %and 100 % aqueous PEG-400 (Sigma); blastoderms remainedin each PEG solution for 30 min. Blastoderms were thentransferred to a 1:1 (v/v) mixture of PEG 400/PEG 1000 at45°C for 30min, followed by PEG 1000 and by PEG 1500 at45°C each for 1 h. Embryos were then embedded in PEG1500 and sectioned shortly after solidification of the PEG.Sections were mounted on slides coated with rubber cement(Lepage's) thinned with ethyl acetate (Drews, 1975) and keptat 4°C until stained. Staining for immunofluorescence wasperformed at room temperature. After treatment for 1 min inacetone sections were washed with three changes, 5 min each,of phosphate-buffered saline (PBS: 150mM-NaCl in 5 mM-Na/K phosphate buffer, pH7-l) . Sections were incubatedfor l h with affinity-purified antibody (8 / lgmr 1 immuno-globulin) or in antiserum diluted 1:400 (15f*gml~' immuno-globulin) both in PBS. Following a PBS wash as outlinedabove, sections were incubated for 1 h in fluorescein-labelledgoat anti-rabbit immunoglobulin (Miles) diluted 1:50 inPBS. After three washes for 5 min each in PBS, slides weremounted in 90% glycerol in PBS with DABCO (Johnson etal. 1982) (Sigma). Controls consisted of sister sectionstreated with preimmune rabbit serum or with commercialrabbit immunoglobulin (Miles). Sections were observed witha Zeiss Photomicroscope III with epifluorescence and photo-graphs were taken with Kodak Ektachrome 200 film.

Results

Antibody purification

The antiserum obtained following the injection sched-ule outlined previously, gave rise to two bands whenreacted against crude lectin extracts in agar diffusiongels (Fig. 1A). When this antiserum was used to probe

nitrocellulose transfers of affinity-purified lectin prep-arations separated on gradient gels two bands wereobtained (Fig. 2a). One band had an approximate Mr

of 6-5X 10'; the second band migrated close to the BSAstandard with a A/r of 70x 103. When this antiserumwas tested against crude extracts of chick blastoderms,it reacted with about 10 different bands (Fig. 3c),while no bands were observed when transfers wereprobed with control preimmune serum. This indicatesthat the rabbit produced antibodies to additional pro-teins that were probably present in undetectable traceamounts in the affinity-purified lectin preparations.The relative staining intensities obtained with therabbit serum probe suggested that L] is present inhigher concentrations in preparations of affinity-puri-fied lectin (Fig. 2a). The molecular weight of L| isclose in range to that reported for other galactose-

• S o -

0-3 0-4 0-5 0-6Diameter of ring (mm)

Fig. 1. A. Agar immunodiffusion of the anti-lectinantiserum used in the experiments. Control preimmuneserum (c) as well as antiserum (as) were tested against thecrude blastoderm extract, which is present in the centrewell of the Petri dish; only a sector of the dish is shown inthis photograph. B. Radial immunodiffusion of theantiserum (AS) and the L| affinity-purified antibody(A.P.Ab). The immunoglobulin standards gave rise to thecircles shown in the upper two rows. The lowest rowshows two repeats of the affinity-purified antibody and twodilutions of the antiserum. C. Shows the standard curveobtained from these experiments.

Galactose lectin expression in the chick blastodenn 485

a b

1 -

2 -

3 -

4 -

5 -6Z

Fig. 2. Immunoblot analysis of purified lectin preparationsand dot-blot analysis of succeeding elutions of L| affinity-purified antibody from nitrocellulose strips. Lanes a, b.Nitrocellulose blots of blastoderm lectin purified on APLcoupled to Affi-Gel 10; the fraction containing the highestlectin activity was electrophoresed for these transfers. Thelectin was run on 10% to 17-5% gradient gels. Lane a.Lectin probed with anti-lectin antiserum diluted l/1000.Lane b. Lectin probed with affinity-purified antibody.Arrows point to LI and LI I. Standards (*Ur) are:1, phosphorylase B, (97-4x10'); 2, BSA (66xlOJ);3, ovalbumin (45X 103); 4, carbonic anhydrase (29X103);5, lysozyme (14-3X103); 6, bovine trypsin inhibitor(6-2X103); 7, insulin b (3-4X103). Lane c. Dot-blotanalysis of three successive elutions of antibody fromnitrocellulose strips containing the blotted Li. The firstthree horizontal rows represent the three successiveelutions of the antibody with 02M-glycine-HC1, pH2-8.The fourth row represents control dots of 5 % BSA, andthe fifth row represents control rabbit serum. Threereplicates are shown for the antibody elution while tworeplicates were done for controls.

binding lectins of tissues from chick embryos (Bar-ondes, 1984). Thus, it was decided to use it as a probeto affinity-purify antibodies using the methodologydescribed by Olmsted (1981) and Talian el al. (1983).Good antibody recovery was obtained with two suc-cessive extractions with glycine-HC1; residual anti-body could also be eluted by a third extraction(Fig. 2c). Other elution protocols were studied. Theinclusion of Tween 20 (Smith & Fisher, 1984) gavereduced resolution due to the fuzziness of the peroxi-dase colour reaction. Extraction with 2% NH4OH in0-5M-NaCl gave results similar to the glycine HC1.Thus three successive glycine -HC1 extractions were

used. The affinity-purified antibodies, reacted withonly a single band when tested against crude blasto-derm extracts and affinity-purified lectin preparations(Figs 3d,e,i, 2b). This was true for nitrocellulosetransfers prepared from 10% gels where L| migratesclose to the tracking dye and from gradient gelstransferred in the presence or absence of methanol(Fig. 3A,B). When estimated from transfers of gradi-ent gels the average .<V/r of L, is 6-5(±f>3)X 10\

Iimminostaiiniig

Having established the identity of blastoderm proteinwith which the affinity-purified antibody reacted, weproceeded to determine the localization of L| in sec-tions of chick blastoderms undergoing gastrulation andearly neurulation. The staining patterns for both areapellucida and area opaca at different developmentalstages are described separately.

In the area pellucida (presumptive embryonic re-gion), the localization of the staining varied with thedevelopmental stage of the embryo. At stage 3 duringearly gastrulation, staining was present in some of thecells of the epiblast anterior and lateral to the nascentimmature primitive streak as well as in some cells ofthis streak. Staining was mainly present in intracellularinclusions (Fig. 4B). At stages 4-5 (definite streak) thefluorescent staining was localized in the lowest portionsof the definite primitive groove as well as in the cellsemerging from this region and migrating laterally toform the endoderm (Fig. 4C,D). In many of thesecells, within the limits of resolution of the technique,staining could be observed at the cell periphery,suggesting an extracellular location. This stainingpattern was specially noticeable in the endodermalcells emerging laterally from the streak (Fig. 4D). Bystage 7 (one-somite stage), staining was restricted tosome endodermal cells (data not shown) and to largeround cells located in the mid-anterior region of theembryo, below the endoderm (Fig. 4E). These cellsprobably correspond to the primordial germ cellsmigrating away from the germinal crescent (Clawson &Domm, 1969; England, 1982). At this stage no stainingwas observed in the neural plate, epiblast and meso-derin. No staining was observed in controls usingadjacent sister sections reacted with immunoglobulinfrom non-immunized rabbits (Fig. 4A).

In the area opaca the pattern of immunofluorescentstaining with the affinity-purified antibodies persistedthroughout the stages examined. At all stages stainingwas absent from the ectodermal cells, while endoder-mal cells showed intense staining (Fig. 5A,B,C,D).Here staining appeared to be mainly cytoplasmic andlocated homogeneously between the numerous yolkplatelets. Intense staining was also present in many ofthe short filopodial extensions of the endodermal cells,as well as in some areas of apposition between these

486 S. E. Zalik el al.

b e d e f

j-ii

LI

A ~ BFig. 3. Immunoblot analyses of Li-specific antibody. Crude lectin preparations were separated on SDS-PAGE slab gelsand transferred to 0-22 j.im nitrocellulose membranes. A. 10% gels; B, 10% to 17-5% linear gradient gels with a 3 %stacking gel (*). Lanes a, g, marker gel slices (not transferred) stained with Coomassie Blue R-250 (swelling of gels relativeto nitrocellulose replicas occurred during staining). Nitrocellulose transfers: lanes b, f, h, stained with 0-1 % Amido Black;lane c, probed with rabbit serum diluted 1/400 in TBS; lanes d, e, i, probed with Li-specific antibody. Gradient gels weretransferred in buffer either with methanol (e, f) or without (h, i). Markers (/V/r) are: 1, ovalbumin (45X103);2, a chymotrypsinogen (25-7X103); 3, j3-lactoglobulin (18-4X 103); 4, lysozyme (14-3X103); 5, bovine trypsin inhibitor(6-2X103); 6, insulin b (3-4X103).

cells and the basal surface of the ectoderm (Fig. 5B);the latter staining pattern was a frequent occurrence insections of this area. It was not possible to assess thedegree of extracellular staining on the endodermal cellsbecause of the intense intracellular staining and theresolution limit of the immunofluorescence techniqueused. In the anterio-lateral area opaca, staining wasalso present in some regions of the leading sheet of themesodcrm that was beginning to penetrate between theectoderm and endoderm; here staining was prominentat the cell periphery, suggesting an extracellular lo-cation (Fig. 5A). Also in the lateral region of the areaopaca (proximal to the area pellucida) distributedamong the endodermal cells, were large round cells thatdisplayed intense intracellular labelling (Fig. 5B,D).These cells probably correspond to the primordialgerm cells of the germinal crescent (Clawson & Domm,1969; England, 1983). In all of the studies reportedhere these staining patterns in the area pellucida andarea opaca were consistent between sister sectionsstained in different experiments as well as betweenembryos fixed with ethanol or paraformaldehyde.However, a better preservation of cellular structure wasobserved in embryos fixed with paraformaldehyde. Atotal of 13 embryos were examined in these studies.

Owing to the limited supply of affinity-purified anti-body, representative sections of different segments ofthe embryo, rather than the complete serial sections ofthe embryo, were examined. In all cases no stainingwas observed when control adjacent sister sections wereallowed to react with commercial immunoglobulinfrom non-immunized rabbits. The same lectin distri-bution was observed when sister sections were stainedwith the lectin antiserum, although background stain-ing was noticeable.

Discussion

Our results using gradient gels indicate that the blasto-derm L[ has a lower relative molecular mass than othergalactose-binding lectins from differentiating tissuesand organs of the chick embryo and from other animaltissues (Barondes, 1984; Zalik & Milos, 1986). Lectinswith small subunits (Mr between 5571 and 6000) have,however, been reported in Vicia faba and Pisiinisativum (Hemperly et al. 1979; Meehan et al. 1982;Quiocho, 1986). We believe L] is a natural polypeptidcand not a breakdown product, since antibodies specificfor Lj do not react with other proteins on nitrocellulosetransfers of crude blastoderm extracts separated on

Galactose lectin expression in the chick blastoderm 487

SDS-PAGE gels. Partial breakdown products reactingwith Li antibodies would be expected as a result ofproteolytic activity. In addition, when the same crudeextracts were run on native gels and transferred tonitrocellulose, the L̂ antibodies reacted with one

distinct band of high molecular weight (Thomson &Zalik, unpublished data).

The iinmunofluorescence studies reported here indi-cate that during early embryogenesis in the chick, L| isexpressed differentially within the cell populations of

488 S. E. Zalik et al.

the blastoderm. In the area opaca no appreciableamount of lectin is associated with the ectoderm, whilethe cells of the EEC as well as the primordial germ cellsdisplay relatively large concentrations of cytoplasmiclectin that persist throughout the stages studied. In thearea pellucida lectin is not detectable in the ectodermlateral to the streak; however, lectin expression occursas cells migrate inwards into the streak and emergelaterally to form the endoderm.

The question arises as to the biological significanceof this lectin in the developmental events that occurduring embryogenesis. Previous studies from this lab-oratory have shown that cell suspensions prepared fromthe area pellucida, as well as from EEC, are highlyagglutinable with Ricimts communis agglutinin, a lectinspecific for j3-D-galactose (Phillips & Zalik, 1982). Thisindicates the glycoconjugates bearing /3-galactosidegroups are present at the surfaces of these cells. It isconceivable, therefore, that the endogenous blasto-derm lectin could interact with these glycoconjugatesand affect a surface-mediated function, i.e. cell ad-hesion. The only cell population in which the effect ofthe endogenous blastoderm lectin on adhesion has beeninvestigated is the EEC. Here, lectin exerts an inhibi-tory effect on cell adhesion that can be overcome bygalactose-bearing compounds (Milos & Zalik, 1982).

Fig. 4. Immunofluorescence staining by affinity-purifiedantibody to LI of transverse sections of primitive streak(stage 4) and one-somite (stage 7) chick embryos.Photographs labelled with the same capital lettercorrespond to the same section observed with phase-contrast (p) or fluorescence (f) optics. In the phase-contrast photographs the background meshwork is thematrix of the rubber cement. A. Control labelled withpreimmune serum diluted 1:400, this is a sister section tosection shown in C-f. B. Section of late stage 3 embryoshowing a very early primitive streak that is not fullymature; staining is restricted to intracellular organelles inthis region. C. Section through a fully developed streak;label is confined to cells at the lowermost portion of thestreak and to the cells emerging from this area (arrows).D. Same section as in C, area slightly lateral to theprimitive streak; no staining is evident in the ectoderm andmost of the mesoderm, label is localized mainly in thelower region of the endodermal cells that faces thesubgerminal cavity (arrows). Staining in this regionappears to be most intense at the cell periphery. Thedirection of the location of the primitive streak is shown byan open arrow. E. Section through the area pellucida of astage 7 embryo showing half of the neural plate with itsadjacent lateral ectoderm. Label is absent from the neuralplate and ectoderm as well as from the mesoderm. Theendodermal cells in this area are also devoid of staining.Many primordial germ cells displaying lectin staining arepresent beneath the endoderm. ec, ectoderm;en, endoderm; g, primordial germ cell; m, mesoderm;np, neural plate; ps, primitive streak. Bar in B, 25,um; barsin the rest, 20,um.

The facts that inhibitory effects of the lectin aretransitory (Milos & Zalik, 1983) and that the spon-taneous release of lectin by EEC is associated withdecreased adhesion (Milos & Zalik, 1982) have led us tosuggest that localized release of this molecule could beinvolved in modulating the transitory adhesions andde-adhesions that occur as cells relocate in the embryo(Zalik & Milos, 1986). In the EEC these rearrange-ments involve the epibolic spreading of cells to formthe yolk sac. Studies from other investigators alsoindicate that EEC from primitive-streak embryos havea ready ability to spread and separate from each other(Sanders et al. 1978; Bellairs, 1982). It is also knownthat lectin activity increases significantly during thespreading of the yolk sac (Mbamalu & Zalik, 1987). Arole in transitory adhesive bond formation has alsobeen postulated for the galactose-binding lectin of somesponges (Miiller & Muller, 1980).

The primitive streak is a structure formed by cells 'intransit' (Bellairs, 1986) from the epiblast to form themesoderm and the embryonic endoderm. Cells in thestreak lose their epithelial appearance and assumea mesenchyme-like shape via the process of de-epithelialization (Bellairs, 1986; Sanders, 1986). Thepresent studies show that when cells move into thestreak they display the lectin as they change fromepithelial-like to fibroblast-like in shape; this lectinbecomes extracellular as cells emerge from the streakand migrate laterally to form the endoderm. Theendogenous lectin could also modulate cell shapeduring the formation of transitory adhesions and de-adhesions that take place in cell ingression. When EECare maintained in stationary culture the blastodermlectin induces the acquisition of a fibroblast-like mor-phology in the epitheloid EEC (Milos & Zalik, 1981). Asimilar phenomenon could occur during epiblast cellingression and emergence from the streak. As theendodermal cells migrate laterally from the streak theydo so in the absence of a visible substratum. Theblastoderm lectin has a strong tendency to aggregate(Zalik et al. 1983; Thomson & Zalik, unpublisheddata). Extracellular aggregates of this molecule couldprovide a transitory substratum over the subgerminalcavity that could be used as scaffold by the spreadingendoderm. A role in cell spreading during metastasishas also been suggested for the endogenous galactose-binding lectins present at the surface of some tumourcell lines (Raz el' al. 1986). The lectin could also bind toother adhesion molecules expressed at this stage, suchas CAMs or cadherins (Edelman, 1985; Hatta &Takeichi, 1986). In the experiments reported here,lectin was found to be expressed transiently in theembryonic endoderm. As mentioned before, owing tothe low amounts of affinity-purified antibody availableonly selected sections of representative regions of theembryo were studied. Detailed studies with serial

Galactose lectin expression in the chick blastodenn 489

sections of complete embryos will be needed to deter-mine if the leetin disappears in all of the regions of theembryonic endoderm.

The primordial germ cells also display abundantleetin in their cytoplasm. It is not clear whether these

cells arise directly in the endoderm or if they arise inthe epiblast and subsequently migrate to the endoderm(England, 1983; Eyal-Giladi et. al. 1981; Ginsburg &Eyal-Giladi, 1986). Regardless of their site of origin,the primordial germ cells will migrate into the area

490 5. E. Zalik et al.

peUucida, penetrate into the developing blood vesselsand gain access to the circulatory system. Sub-sequently, in development the primordial germ cellsmigrate out of the blood vessels to colonize the gonad(England, 1983). The endogenous lectin in these cellscould be associated with the cytoplasmic inclusions thatstain with periodic acid—Schiff (Clawson & Domm,1969; Fujimoto el al. 1976) and with Ricinus coiiiiiiiinisagglutinin (Didier el al. 1981). The primordial germcells could externalize the lectin and use it during theirmigration to and from the blood vessels to colonize thegonad.

Because of the very high concentration of intracellu-lar lectin present in the EEC and primordial germ cells,it is not possible to ascribe with certainty a cell surface

Fig. 5. Immunofluorescence staining by affinity-purifiedantibody to LI of transverse sections of primitive streak(stage 4) and one-somite (stage 7) chick embryos. Thisplate shows the staining pattern of the area opaca.Photographs labelled with the same letter represent thesame section observed with phase-contrast (p) orfluorescence (I) optics. A. Transverse section through theanterior region ot a stage 4 embryo showing to the left theregion of transition between the area pellucida and thearea opaca; the direction of the location of the areapellucida is shown by an open arrow. In this region of theembryo the mcsodermal layer has started to penetratethrough the area opaca. The peripheral staining on thecells forming the leading edge of the mesodermal sheet isshown (arrows). Cells of the extra-embryonic endodermstain brightly; here staining is intracellular around andbetween the yolk granules as well as in the peripheralcytoplasm. Some lobopodial extensions of the cells of theextra-embryonic endoderm are also stained (#). B. Sectionshowing the lateral area opaca close to the end of theregion of the germinal crescent of a stage 4 embryo. In thisregion the cells of the extra-embryonic endoderm are stillin contact with the ectoderm. A blunt filopodial extension(*) of these cells that is in contact with the ectoderm stainsbrightly with this antibody; this was observed frequently.The lectin also appears to be deposited on the basal regionsof the ectodcrmal cells (arrows); some primordial germcells are also stained. C. Transition between area pellucidaand area opaca of a stage 7 embryo. The direction of thelocation of the area pellucida is shown by an open arrow.The lack of staining of the ectoderm as well as the increasein the number of stained endodermal cells as oneprogresses laterally through the area opaca is evident.Staining in these latter cells is mainly cytoplasmic.D. Section through the germinal crescent of a stage 7embryo. Ectodcrmal cells are not stained. Intense stainingis present in the primordial germ cells. The intensestaining of these cells overshadows that of the cells of theextra-embryonic endoderm. A pseudopodial extensionsimilar to that described by England (1983), in primordialgerm cells is heavily stained (#). ap, area pellucida;ec, ectoderm; en, endoderm; een, extra-embryonicendoderm; g, primordial germ cells; in, mesoderm. Bars,2G>n.

location for this lectin. Studies at the electron-micro-scope level arc needed to determine the localization ofthis lectin in the above cells as well as in those of theprimitive streak.

This work was supported by the Medical Research Councilof Canada and by the National Science and EngineeringResearch Council of Canada. We thank Miss Julie Scheinasfor typing the manuscript.

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{Received I June 19S7 - Accepted 24 July 1987)

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