Roux's Arch Dev Biol (1986) 195:265-275 Roux's Archives of Developmental Biology �9 Springer-Verlag 1986

The presence of an extracellular matrix between cells involved in the determination of the mesoderm bands in embryos of Patella vulgata (Mollusca, gastropoda)

Wiel M. Kiihtreiber, Jos van der Bent, Adrie W.C. Dorresteijn, Arjan de Graaf, Jo A.M. van den Biggelaar, and Cees A.M. van Dongen Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3508 TB Utrecht, The Netherlands

Summary. In 32-cell stage embryos of Patella vulgata one of the macromeres contacts the animal micromeres, and as a result is induced to differentiate' into the stem cell of the mesodermal cell line. In this study we show the pres- ence of an extracellular matrix (ECM) between these two interacting cell types. The ECM appears to be formed by the micromeres during the 32-cell stage. Staining experi- ments with alcian blue and tannic acid indicate that it con- tains glycoconjugates, possibly in the form of proteogly- cans. The characteristics of the ECM were examined further by fluorescein isothiocyanate (FITC)-lectin labelling. Of 17 leetins tested, concanavalin A (ConA), succinyl-ConA, LCH-B (Lens culinaris) and PEA (Pisum sativum) showed a positive labelling of the ECM. These results are in accor- dance with the electron microscopic data. The appearance of the ECM at this specific stage and place suggests that it might play an important role in the induction of the mesodermal cell line.

Key words: Extracellular matrix - Mollusca - Mesoderm determination - Lectins - Cell contacts

Introduction

Up to the 32-cell stage, embryos of the marine gastropod Patella vulgata consist of four equal quadrants of cells ar- ranged in a radially symmetrical pattern around the animal- vegetal axis. From cell lineage studies, it is known that during further development of Patella embryos, as in em- bryos of other equally cleaving molluscs, each quadrant develops according to a specific pattern and gives rise to a well-defined part of the larva (Verdonk and van den Big- gelaar 1983). The D-quadrant produces the main part of the embryo. It will develop the stem cells of most of the adult ectoderm and of the adult mesoderm.

During P. vulgata embryogenesis, the interval between fifth and sixth cleavage is prolonged for about 1 h longer than the intervals between earlier cleavages. During this phase the four vegetal macromeres and the animal micro- meres elongate and stretch towards each other. Eventually one macromere (designated 3D) will acquire and maintain a firm contact with the animal micromeres and establish a central position within the embryo, thereby shifting the radial symmetry into a bilateral one (van den Biggelaar 1977). Up to the 60-cell stage, divisions of all corresponding

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cells of the four quadrants take place synchronously; how- ever, the completion of the sixth cleavage cycle is asynchro- nous. The centralized 3D will divide significantly later than the other three macromeres. In Patella, as in other molluscs, this central macromere will play a key role in the dorsoven- tral differentiation of the developmental pattern within the embryo as a whole (Clement 1962; van den Biggelaar and Guerrier/979).

It has been shown that if the contact between the macro- meres and the micromeres is prevented by either dissocia- tion or cell deletion (van den Biggelaar and Guerrier 1979; Arnolds et al. 1983) or by cytochalasin-B treatment (Mar- tindale et al. 1985), none of the macromeres will develop into a mesentoblast. Therefore, it is assumed that in equally cleaving molluscan eggs the specific properties of the central 3D macromere are the result of interactions between this macromere and the micromeres during the interval between the fifth and sixth cleavage.

This aspect of the early development of equally cleaving molluscs has much in common with the differentiation be- tween the inner and outer cells in the mouse embryo (Gra- ham and Lehtonen 1979). Both systems demonstrate the importance of cellular interactions in embryonic develop- ment. We are now analysing the character of the cellular interactions involved in the determination of the mesoder- mal stem cell. In general, one may assume four possible mechanisms for signal transduction (Grobstein 1955): (1) the formation of intercellular communication channels; (2) diffusion of signal molecules from one cell to another over relatively long distances; (3) indirect contact by an extracel- lular matrix; and (4) a direct contact between ligands and receptors in two adjacent cell membranes. In this paper we present electron microscopical evidence for the presence of an extracellular layer between the inducing animal micro- meres and the future stem cell of the mesoderm bands.

Extracellular layers in developing embryos generally contain glycoproteins and proteoglycans (for reviews, see Farquhar et al. 1982; Hascall and Hascall 1981). For sever- al of these components a morphogenetic role has been sug- gested (e.g. Bissell et al. 1982; Spicer et al. 1981; Toole 1981; Yamada 1983). Since lectins bind to specific sacchar- ide configurations (for reviews, see Brown and Hunt 1978; Roth 1978), they are particularly well suited for histochemi- cal investigations on the molecular characteristics of extra- cellular layers (Bee 1982; Bissell 1982; Brabec et al. 1980; Nemanic et al. 1983; Reano et al. 1982; Wu et al. 1983). Therefore, fluorescein isothiocyanate (FITC)-lectins were

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used for the partial characterization of the extracellular ma- trix of 32-cell stage Patella embryos.

Materials and methods

Embryos. Adult specimens of P. vulgata were collected on the French Atlantic coast at Roscoff, Ambleteuse or Dieppe. They were kept in tanks with recirculating filtered sea water. Mature gametes were obtained by dissecting out the gonads from adult animals. Artificial fertilization and synchronization were performed according to the method of van den Biggelaar (1977). Embryos were cultured at 19 ~ C and collected at successive stages.

Electron microscopy. For electron microscopical investiga- tions, embryos of the appropriate stages were washed in acidified sea-water (pH 4.0) to remove the gelatinous egg- capsule and then fixed for 2 h at room temperature in 2.5%-4% glutaraldehyde (TAAB Laboratories Equipment Ltd. Berkshire, England) in either 0.1 M phosphate buffer (pH 7.2) or 0.1 M cacodylate buffer (pH 7.2) supplemented with 0.2 M saccharose. In some experiments embryos were partially dissociated in Ca ++ and Mg++-free sea-water (CMFSW) prior to fixation. Tannic acid or alcian blue 8 GX was added to the fixation mixture (1% end-concen- tration) to stabilize the spatial configuration of the extracel- lular matrix (ECM). After several washes in buffer, the embryos were post-fixed for 1 h in either 1% or 2% OsO4 in cacodylate buffer (pH 7.2). After fixation the embryos were washed and orientated in 2% agar to enable sectioning parallel to the animal-vegetal axis (van der Wall and Doh- men 1978). Next they were dehydrated in a graded series of ethanol and embedded in LADD LX-112 resin (Ladd Research Industries Inc. Burlington, USA) via propylene oxide.

Ultrathin sections parallel to the animal-vegetal egg axis were made on a Reichert OM U3 ultramicrotome, and mounted on copper one-hole grids supported by a piolo- form film. Some sections were post-stained with uranyl ace- tate and lead citrate according to Reynolds (1963). The preparations were studied with a Philips EM 300.

Identification of cells. For three-dimensional reconstruction of embryos, semi-thin serial sections (0.5 gm) parallel to the animal-vegetal egg axis were stained with a mixture of 1% methylene blue in 1% borax and 1% azure II (1:1 v/v). Reconstructed embryos allow the identification and denomination of all blastomeres because of their fixed rela- tive positions in the embryo.

Lectins. Fluorescein isothyocyanate (FITC)-conjugated lec- tins were purchased from several companies: BPA, BSA-I, BSA-II, HPA, LBA, MPA, PNA, SJA and WFA from Polysciences Inc. (Northampton, UK); ConA, PMA-L and UEA-I from E-Y Laboratories (San Mateo, USA); LCH-B, SBA and WGA from Miles Yeda Ltd. (Rehovot, Israel); PEA and succinyl-Con A from Sigma Chemical Company (St. Louis, USA). These substances were reported by their manufacturers to be free of unbound FITC (for abbrevia- tions, see Table I).

Procedure for paraffin embedding. Selected embryos were fixed overnight at 4~ with 4% (w/v) paraformaldehyde

in phosphate-buffered solution (PBS) (0.15 M NaC1 in 0.1 M phosphate buffer, pH 7.4) or with Bouin's fixation mixture (15 ml water-saturated picric acid, 5 ml 40% form- aldehyde and 1 ml glacial acetic acid). After several washes in PBS the embryos were quenched for 10 min with 50 mM NHgC1 in PBS to reduce autofluorescence, and washed again with PBS. Next they were orientated individually in 2% agar in distilled water to enable sectioning parallel to the animal-vegetal axis (van der Wall and Dohmen 1978). The orientated embryos were dehydrated in a graded series of ethanol, processed into xylene and embedded in paraffin (melting point 57 ~ C) at 60 ~ C. Sections (4-6 gm) were mounted on protein-glycerin-coated glass slides and air- dried for at least 2 days at 6 ~ C. Mounted sections were deparaffinized in 100% xylene (two steps of 3 min each) and rehydrated. They were then washed in PBS and treated with 3 M urea in PBS for 5 rain according to Hausen and Dreyer (1982). After a last wash in PBS, the sections were ready for lectin labelling.

Procedure for cryo-embedding. Selected embryos were fixed for 2 h at 4 ~ C with either 4% paraformaldehyde in 0.1 M PBS (pH 7.4) or with Bouin's fixation mixture. After several washes in PBS, they were embedded without orientation in agar (1% in distilled water) in groups of approximately 50 embryos each. Embedding in acrylamide was performed according to Hartmann (1984) with some changes. The em- bedding mixture was prepared by mixing 20 ml 3.398 M acrylamide (Serva Heidelberg, FRG, recrystallized accord- ing to Loening 1967) containing 0.048 M N, N' methyleneb- isacrylamide (Biorad Utrecht, The Netherlands), 10ml 0.2 M S6erensen phosphate buffer (pH 7.2), 35 ml gelatine (16% w/v), 20 ml Tissue Tek (Miles Yeda Ltd. Rehovot, Israel), and 0.2 ml TEMED (N,N,N',N' tetramethylethy- lenediamine, Merck Amsterdam, The Netherlands). Each group was preincubated in 5 ml of the embedding mixture for I h at 37 ~ C, and then embedded in I ml of fresh embed- ding mixture and polymerized by adding 17 gl 2% ammoni- um persulphate (Biorad) and fixed with 45 gl fixative (10% w/v formaldehyde and 6.25% w/v glutaraldehyde in 0.1 M phosphate buffer pH 7.2). Next the embedded embryos were cryo-protected by subsequent incubations in 10% di- methyl sulphoxide (DMSO) in PBS for 1 h at room temper- ature, and 1 M sucrose in distilled water overnight at 4 ~ C. The next day the preparations were embedded in Tissue Tek and frozen in expanding COg. Cryo-sections (4-7 gin), made at - 2 5 ~ in a Bright cryostate, were mounted on poly-L-lysine-coated glass slides and allowed to dry for 20 min at room temperature.

Lectin labelling. FITC-lectin working-solutions were pre- pared by 1 : 200 dilution from the purchased stock-solutions with PBS containing MnC12 (0.1 mM) and CaCI2 (0.1 mM). Mounted paraffin or cryo-sections were quenched for 10 min with 50 mM NH4C1 and washed in PBS~ Next the sections were incubated in the dark with 20 gl lectin solu- tion for 2 h at 37 ~ C, or overnight at room temperature. After labelling they were washed in PBS (2 x 10 min) and distilled water (10 min), and mounted in PBS-buffered gly- cerol containing 1 mg/ml p-phenylenediamine to reduce bleaching (Johnson and de C. Noguira Aranjo 1981). Slides were examined with a Zeiss epifluorescence microscope (ex- citation filter: 490 nm; barrier filter: 528 nm). Photomicro- graphs were taken on Kodak Tri-X ASA 400 film.

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Tests for labelling specificity were performed by incu- bating sections with mixtures of FITC lectins that showed positive labelling on the ECM and their specific sugars at end-concentrations ranging from 0.1 M to 0.3 M. In addi- tion, control experiments were performed, in which the sec- tions were pre-incubated for 30 min at room temperature in BSA (5% in PBS) prior to lectin/sugar incubation.

Results

Electron microscopy

Embryos were fixed at successive intervals between 16-cell (fourth cleavage) and 64-cell (sixth cleavage) stages, and investigated at the ultrastructural level.

The 16-cell stage. At the 16-cell stage the intra-embryonal cell surfaces of both micromeres and macromeres were smooth, and did not carry extracellular material associated with the cell membranes. Patches of alcian-blue-positive material irregularly distributed between the blastomeres were sometimes observed in partially dissociated embryos.

At 20 min afterfzfth cleavage. At this stage there was still a cleavage cavity, and the vegetal macromeres were not

.yet in contact with the micromeres of the opposite animal pole. In all embryos examined a considerable amount of extracellular material was found in the cleavage cavity. It appeared in the form of a meshwork of fibrillar material (Fig. 1). In particular, the derivatives of the first-quartet micromeres in the animal hemisphere showed a distinct layer of extracellular material along the centrally located cell tips, although minor spots of ECM were also present along the tips of other micromeres. This layer had the ap- pearance of a basal lamina. It consisted of an electron- lucent layer directly facing the cell membrane and an elec- tron-dense layer adjacent to it. The layers were comparable to a lamina tara and a lamina densa, respectively (Hascall and Hascall 1981). Frequently, electron-dense strands were found, radiating from the lamina-densa-like layer to the cell membrane (Fig. 1 b).

The overall thickness of the extracellular layer varied between 30 and 40 nm. Extracellular material was found dispersed throughout the cleavage cavity and appeared to be connected to the extracellular layer (Fig. I).

Many large vesicles with unit membranes were present along the centrally directed cell tips of the animal micro- meres (Fig. 1). These vesicles resembled secretion vesicles, as described by Hascall and Hascall (1981). At some places these vesicles were closely opposed to the cell membrane, suggesting that their contents might be exteriorized (Fig. I a, d).

At 45-70 rain after fifth cleavage. At this stage all four mac- romeres stretched far into the cleavage cavity and met the inner cell tips of the animal micromeres. The extracellular layer could be observed on the animal micromeres, whereas little or no material was found on the vegetal macrorneres (Fig. 2a, b). The remnant of the cleavage cavity was still filled with a coarse meshwork of extraceUular fibrillar mate- rial, which seemed to be shed from the extracellular layer on the micromeres (Fig. 2c, d).

The presumed secretion vesicles in the first-quartet mi- cromeres were observed in some embryos, whereas in others

the micromeres were empty (Fig. 2). This observation strengthens the idea of formation of the extracellular layer, and eventually also the meshwork of extracellular material in the cleavage cavity, by extrusion of this material by the presumed secretion vesicles.

At 90 min after fifth cleavage. In embryos of this stage, one of the macromeres (3D) had attained a central position and was the only macromere that contacted the overlying micromeres. Only small intercellular clefts were left of the cleavage cavity. The extracellular layer could be clearly dis- tinguished between the tip of the central 3D macromere and the animal micromeres (Fig. 3). The layer of extracellu- lar substance was mainly found at the central tips of the micromeres.

The structure of the extracellular material appeared to be very sensitive to the kind of fixation used. The structure of the ECM was enhanced when fixation was performed in the presence of alcian blue or tannic acid. In addition, when tannic acid was used, the extracellular layer had the appearance of a very electron-dense layer directly opposed to the cell membrane and a coarse granular layer at a short distance from it (Fig. 2a, b, d). These granules had the same size (approx. 30 nm) and looked like proteoglycan monomers as observed by Takagi et al. (1983) in epiphyseal cartilage and like the proteoglycan granules in the basement membrane of sea urchin embryos (Spiegel et al. 1983).

Lectin labelling

All FITC-conjugated lectins were tested on 4- to 6-gin sec- tions parallel to the animal-vegetal egg axis, shortly before or at the contact stage between 3D and the animal micro- meres (about 45 min and 60 rain after 5th cleavage, respec- tively). Of the 17 lectins that were tested on sections of about 70 embryos, only 4 showed positive labelling of the ECM (See Table l, Fig. 4). All lectins, with the exception of PHA-L, gave a heavy labelling of the egg capsule (the acellular layer that can be seen surrounding the egg after it has stripped of its chorion; Fig. 4e). Therefore, the egg capsule was usually not removed, as it was useful as an internal control for the labelling procedures.

Shortly before the interaction stage, there was still a small cleavage cavity left. In the sections a large part of this cavity was filled with a rather dense material, which showed positive labelling with ConA, succinyl-ConA, PEA and LCH-B. In the majority of the preparations, the label- ling of this blastocoelic material was most intense in the vicinity of the tips of the micromeres (Fig. 4a, c). Often the outlines of the apex of the vegetal cells and of the blasto- coelic material were approximately complementary (Fig. 4a, c, f). We assume that the empty space facing the macromeres is an artefact due to shrinkage effects caused by one or more steps of the embedding procedure: collaps- ing of the highly hydrated spatial configuration of ECM (in particular of proteoglycans) upon dehydration is a well- known phenomenon (see Singley and Solursh 1980). This is consistent with the observations of positively labelled fi- brillar extensions of the blastocoelic material, which stretch towards (and contact the tips of) the macromeres (Fig. 4b, c).

Control incubations were performed for those four lec- tins that showed positive labelling of blastocoelic material. In these experiments, the lectins were mixed with 0.1-0.3 M

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Fig. I a-d. Ultrathin sections of Patella vulgata embryos approximately 20 min after fifth cleavage. An extracellular layer adjacent to the centrally located membranes of the micromeres is clearly visible, a, d Arrows indicate vesicles resembling secretion vesicles as described by Hascall and Hascall (1981). b Arrows indicate electron-dense strands, radiating from the cell membrane towards the outer border of the extracellular layer, c The cleavage cavity is filled with a considerable amount of fibriUar material resembling proteoglycans (arrow). The embryos in a-e were prefixed with glutaraldehyde only; in d prefixation was performed with a mixture of glutaraldehyde and alcian blue. cc cleavage cavity; y yolk granule. Bars represent 0.1 ~tm

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Fig. 2a--d. Ul t ra thin sections of P. vulgata embryos approximately 45-70 min after fifth cleavage (a 60 min, b 60 rain, c 70 min and d 50 min). An extracellular layer is found on the micromeres, whereas the macromeres are relatively devoid of extracellular material. In some embryos excretion vesicles can still be found in the micromeres (arrows in a), whereas others are empty (b). Arrows in e and d indicate shedding of material from the extracellular layer into the blastocoelic cavity. All preparations were prefixed with a mixture of glutaraldehyde and tannic acid. cc cleavage cavity; M macromere; rn micromere; y yolk granule. Bars represent 0.1 gm

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Fig. 3a, b. P. vulgata embryos approximately 90 min after fifth cleavage, a Of the cleavage cavity only small intercellular clefts are left, in which extracellular material still can be distinguished. ECM material is still visible on the micromeres (arrow). b After contact between the 3D macromere and the micromeres is established, the extracellular layer can still be distinguished between them (arrow). All preparations were prefixed with glutaraldehyde only. M 3D macromere; m micromere; mi mitochondrium. Bar in a represents 1 gm, in b 0.1 gm

of the sugars 0~-D-mannose and 1-O-methyl-~-D-glucopyr- anoside (Sigma), which are specific for the positive lectins (cf. Table 1). Fluorescence decreased in correlation with the sugar concentration. At 0.1 M there was still a substantial amount of fluorescence left, whereas at 0.3 M the staining intensity reached the level of autofluorescence, which in quenched preparations was negligible.

The appearance and labelling intensity of the blasto- coelic material in Bouin-fixed embryos was superior to paraformaldehyde-fixed material, although after Bouin-fix- ation most of the egg capsule was lost, probably due to

the low pH of the fixation mixture. Fixat ion with parafor- maldehyde resulted in good preservation and high labelling intensity of the egg-capsule (Fig. 4e), but in most cases the major part of the blastocoelic material was lost (Fig. 4d), leaving only a thin layer of ECM on the inner cell tips of the micromeres. This remaining ECM had good labelling properties (Fig. 4d). At the stage where the 3D macromere was seen in contact with the animal micromeres, only a thin layer of extracellular material was present between these two cell types. This ECM had the same labelling char- acteristics as at the earlier stages (Fig. 4h).

Fig. 4a-h. Sections of 32-cell stage P. vulgata embryos incubated with FITC-conjugated lectins. All sections except g were made approxi- mately parallel to the animal-vegetal egg axis; the animal pole is indicated by the small arrow head. The embryos in a-e, f, g were fixed in Bouin; in d, e, h paraforrnaldehyde was used. a, b ConA-labelling of paraffin section (a) and of cryo-section (b). The blastocoelic material is heavily stained. Note the complementary shape of this material and the tips of the vegetal cells in a. c LCH-B labelling of cryo-section. A substantial amount of cytoplasmic labelling is found, whereas nuclei are negative. The blastocoelic material is heavily labelled. Note the fibrillar protrusions, which can be seen in contact with the tips of the macromeres (arrow). d LCH-B labelling of paraffin section. Only a thin layer of positively labelled extracellular material is left in the blastocoelic cavity (arrow). The embryo was decapsulated by incubation for 3 min in acidified sea-water (pH 4.0) prior to fixation for a more accurate integral measurement of the exposure time. e ConA labelling of paraffin section. The egg-capsule was not removed. Note the very high labelling of the capsular material, f, g ConA labelling of cryo-sections. Note the fluorescence in the tips of the vegetal cells in f. The central core of fluorescence is also visible when the plane of sectioning is approximately perpendicular to the animal-vegetal egg axis (g). h ConA labelling of paraffin section. When contact is established, only a thin layer of positively labelled material (arrow) is left between the micromeres and the 3D macromere (outlined). Magnification bars represent 20 ~tm

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Table 1. Labelling of the extracellular matrix and of the capsule of 32-cell stage Patella vulgata embryos by the 17 lectins tested as assessed by visual examination

Lectin Source Main sugar specificities Labeling on

ECM Capsule

BPA Bauhinia purpurea D-Galactose, e-D-GalNac - + + BSA-I Bandeira simplicifolia e-D-Galactose -- + + BSA II Bandeira simplicifolia e-D-Galactose -- + + ConA Canavalia ensiformis e-D-Glucose, e-D-mannose + + + + + HPA Helix pomatia C+D-GalNac - + + LBA Phaseolus lunatus D-Gal, 7-D-GalNac - + + LcH-B Lens culinaris ~-D-Glucose, e-D-mannose + + + + MPA Maclura pomifera e-D-Galactose, C+D-GalNac - + + PNA Arachis hypogaea D-Gal-fl(1-3)-GalNac - + + PEA Pisum sativum a-n-Glucose, e-D-mannose + + + PHA-L Phaseolus vulgaris ~-d-GalNac - - SBA Soybean/Glyeine max D-Gal, e-D-GalNac - + + SJA Sophorajaponica C+D-GalNac - + + SuccinylConA Canavalia ensiformis ~-n-Glucose, e-D-mannose + + + + + UEA-I Ulex europaeus c~-L-Fucose -- + + WFA Wistariafloribunda e-D-GalNac - + + WGA Wheat germ/Tritieum vulgate (N-Acetyl-D-glucosamine), -- + +

~-D-GalNac= N-Acetyl-~-D-galactosamine; - = no labelling; + = slightly positive, + + = positive; + + + = strongly positive labelling

A differential labelling of the cytoplasm of both micro- meres and macromeres was observed, especially in cryo- prepara t ions (Fig. 4c, f), whereas nuclei were not labelled. In a number of cases, the tips o f the vegetal cells, especially of the macromeres , showed relatively high labelling (Fig. 40 . This labelling pa t te rn was par t icular ly clear when the plane of sectioning was perpendicular to the egg axis at the level o f the macromeres (Fig. 4 g).

Discussion

In this study we were interested in the nature of the cell contacts between the animal blastomeres descended from the first quar te t of micromeres and the central macromere f rom which the mesodermal bands will develop. Cell dele- t ion experiments have shown that this contact is essential for the induct ion of the mesodermal cell line (van den Bigge- laar and Guerr ie r 1979; Arnolds et al. 1983). In this s tudy we show that an E C M is present between the involved cell types. The 32-cell embryo of Patella produces two types of E C M : (1) a three-dimensional meshwork o f E C M fibres and (2) a dist inct extracellular coat with the appearance of a basal lamina as found in other tissues (Gordon and Bernfield 1980; Fu r thmayer et al. 1982; Hascal l and Has- call 1981).

After g lutara ldehyde/ tannic acid fixation, electron- dense mater ia l is found in the lamina-rara- l ike layer, which appears to be associated with the outer leaflet of the cell membrane. This mater ia l looks similar to the dense thicken- ings (plaques) against the extracellular surface of the mam- mary epithelia as described by G o r d o n and Bernfield (1980). Par t ia l dissociat ion is necessary for sufficient pene- t ra t ion o f tannic acid into the blastocoelic cavity. However, we do not think that this t reatment has a negative effect on the integri ty of the ECM. When the total amounts of E C M with and without par t ia l dissociat ion are compared, there seems to be no clear difference; with tannic acid the ul trastructure is even enhanced. Apparent ly , f ixation with

tannic acid leads to the preservat ion of one or more extra components in the lamina-rara- l ike layer. Part icular ly acidic glycosaminoglycans ( G A G ) are lost by the rout ine method of fixation with glutaraldehyde only, but they are preserved if tannic acid is included (Takagi et al. 1983). The presence of the electron-dense layer adjacent to the outer surface of the cell membrane after the addi t ion of the mordan t tannic acid represents G A G (Sannes et al. 1978; Singley and Solursh 1980; Spicer et al. 1981; Toole 1981). The presence of G A G may also be inferred from the results after f ixation in the presence of alcian blue (Harr- isson et al. 1985; Schenk 1981; Quintarelli et al. 1964). Also, the lamina-densa-l ike layer looks different after the addi t ion of tannic acid to the fixative, having a granular appearance. The diameter o f the granules is about 30 nm, which is in the order o f pro teoglycan monomers (Takagi et al. 1983).

The E C M is formed directly preceding contact between the animal micromeres and the centralized macromere 3 D. I t is presumed to be produced by the mesoderm inducing micromeres, as in the early 32-cell stage their tips especially are rich in vesicles which progressively diminish (Fig. 1, 2), a l though there seems to be some var ia t ion in the time of d isappearance of the vesicles in the micromeres of differ- ent embryos (cf. Fig. 2a, b, d). These vesicles have the ap- pearance of secretion vesicles as described by Hascall and Hascal l (1981). Apa r t from close membrane apposi t ion of these vesicles (Fig. I a, d), we found only few indicat ions for exocytosis; the presence of small vesicles in the blasto- coelic cavity also seems strange (Fig. 2c). However, at pres- ent there is no other logical explanat ion for secretion of E C M mater ia l into the blastocoelic cavity. Exocytosis might either be very stage-specific or insufficiently preserved by the fixation procedures used.

Lectin staining

As ment ioned above, the results o f the EM study indicate that the E C M contains glycoconjugates, possibly in the

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form of proteoglycans. The results of the lectin labelling experiments provide further evidence for this (see Table 1). Positive labelling of the ECM with (succinyl-) ConA, LCH- B and PEA indicates the presence of glucose and maunose residues as terminal groups (see Roth 1978). Glycoproteins with these sugars in terminal positions are commonly found in extracellular material, including basal laminae, indicating their highly conserved nature (for reviews, see Hay 1981; Bissell et al. 1982). Specific labelling of blastocoelic material by the four FITC lectins may therefore indicate the presence of at least some of the components that are commonly found in extracellular layers. Immunocytochemical tech- niques will be used in future investigations to determine which components are present in the 32-cell stage ECM of Patella.

The positive labelling of the egg-capsule obtained with all lectins, with the exception of PHA-L, indicates that the incubation conditions were not restrictive. The observation that only those lectins which are specific for glucose or mannose groups exhibit positive labelling of the blastocoelic material does not necessarily imply, however, that other terminal sugars are not present (Table 1). Labelling may be negatively influenced by charge distribution and steric hindrance has also been reported to play a role in the speci- ficity of lectin binding (Brown and Hunt 1978). The nega- tive results for the other lectins are thus not completely conclusive.

ConA has a higher affinity for mannose and glucose residues than LCH-B or PEA (Roth 1978). This probably explains the stronger labelling intensity of (succinyl-) ConA as compared to the other lectins, although this might also be related to differences in the amount of FITC which is conjugated to the respective lectins. PHA-L does not label the egg capsule, whereas other lectins with the same sugar specificity show positive labelling. This is a phenomenon commonly found for this lectin: PHA recognizes its specific saccharides only in particular configurations (Spengler and

Weber 1981). The electron microscopic data show that the blastocoelic

material consists of two distinct areas: a relatively dense layer of 40-50 nm against the micromere tips and a more loose meshwork of fibrillar material throughout the blasto- coelic cavity. The present results show that the fluorescence pattern after labelling with (succinyl-) ConA, LCH-B or PEA corresponds to these areas. In the majority of prepara- tions, the level of fluorescence near the tips of the micro- meres is higher than in the rest of the blastocoelic cavity. This probably reflects the more dense structure of the extra- cellular material at the tips of the micromeres. As the ECM is probably excreted by the micromeres, the concentration of ECM components at the micromere cell surface may simply be higher. However, it cannot be excluded that the difference in labelling intensity is due to differences in the molecular composition of both types of blastocoelic materi- al. Steric hindrance might also be of importance in this respect (Harrisson et al. 1985).

The sugar concentrations which were found to be neces- sary for efficient competition in our control experiments were relatively high as compared with the concentrations used by other authors. In the literature, a concentration of 0.1-0.2 M is usually mentioned (see, e.g., Reano et al. 1982; Roth 1978), whereas we obtained complete competi- tion when 0.3 M was used, in which case only a neglegible amount of background fluorescence was left. Nevertheless,

we are confident that the observed labelling is specific and not due to aspecific binding. This conclusion is strengthened by the observation that preincubation in 5% BSA before FITC-lectin labelling did not result in a lower observable level of fluorescence.

The preservation of blastocoelic material at the light microscopical level appears to depend on the kind of fixa- tive used. Paraformaldehyde fixation results in a poor pres- ervation of the blastocoelic material and in a good preserva- tion of the egg-capsule, whereas opposite results are ob- tained when Bouin is used. Apparently, the positively react- ing 32-cell stage ECM components are not as readily ex- tracted at low pH as are capsular components. Cytoplasmic labelling is probably mainly due to the high amount of yolk material, as this is rich in glycoproteins (van Dongen and Goedemans, unpublished work). However, the relative- ly heavy labelling which is found in many preparations at the apex of the macromeres remains to be explained (Fig. I b, f, g).

In the Introduction, four possible mechanisms were mentioned for the transduction of the mesoderm-inducing signal from the animal micromeres to the central macro- mere. The results of earlier dye-coupling experiments (Dor- resteijn et al. 1983) and EM observations (Dorresteijn et a l . 1982) make it very Unlikely that the signal is transduced through gap junctions: although in embryos of Patella the fluorescent marker Lucifer yellow is easily transported from cell to cell after fifth cleavage, no direct transport has been observed between the central macromere 3D and the animal micromeres (Dorresteijn et al. 1983). In an electron micro- scopic study of this intercellular contact no gap junctions were found (Dorresteijn et al. 1982). Both results are not in favour of signal transduction by gap junctions.

The second possibility mentioned was diffusion of signal molecules over relatively long distances. This mechanism is also unlikely, as the diffusible molecules would not only reach the central macromere but also the other three macro- meres. As a consequence, all four macromeres would be induced to develop mesoderm. This, however, does not oc- cur.

The remaining two possibilities refer to a direct or indi- rect contact between the involved cells as a prerequisite for signal transduction. The presented results demonstrate that an extracellular layer has been found specifically be- tween the first-quartet micromeres and the mesoderm-form- ing central macromere. The specific localization of this bas- al-lamina-like structure is in full harmony with the pre- viously assumed way in which one out of four equipotential macromeres is induced to produce the mesodermal cell line. After removal of the first-quartet cells, thus in the absence of an extracellnlar layer, the induction fails to occur (van den Biggelaar and Guerrier 1979).

Nothing definite can be said about the role of the layer of ECM at the inner side of the micromeres, nor of the meshwork of ECM present in the cleavage cavity. However, two points may indicate a possible role: first, the presence of the ECM surface coat, especially on the tips of the induc- ing micromeres, and its position between the two types of cells involved; second, its formation immediately preceding the induction of the mesoderm.

The way in which the ECM coat plays a role is still unknown. A possibility might be the development of mutual adhesiveness between the two interacting cell types and sta- bilization of the mutual contacts. This would allow direct

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contact between the cell membranes and finally a receptor - ligand binding. ECM components might also play an inducing role. In the literature, examples supporting these possibilities are described. Models in which the ECM is assumed to exert its effects via the cytoskeleton and the nuclear matrix have been described by Bissell et at. (1982) and Yamada (1983). Examples of the ligand - receptor model for the interaction of adjacent cell membranes can be found in the article by Bluemink et at. (1976).

Because of the very rigid three-dimensional configura- tion of the cells in a 32-cell-stage Patella embryo, as well as the presence of the ECM on the intra-embryonal cell surfaces of the micromeres, the possibility should be consid- ered that the ECM might not only play a possible role in the "anchorage" of 3D, but might also play a more direct role in the induct ion process, by supplying the induc- t ion signal itself, or by regulating it (Farquhar et al. 1982; Bissell et al. 1982).

In summary, we may conclude that the animal micro- meres produce an extracellular layer that probably contains proteoglycans and is very similar to the general structure of a basal lamina. Only the centralized macromere comes into contact with this extracellular layer and it is assumed that this process induces the central macromere 3D to pro- duce the mesodermal bands. In future experiments, the four lectins that react positively with the ECM material will be used as experimental probes in an attempt to investigate in more detail the role of the ECM in the inductive cellular interactions that take place during the 32-cell stage.

Acknowledgements. The authors wish to express their gratitude to Prof. N.H. Verdonk for reading the manuscript critically and for stimulating suggestions. B. Wagemaker is thanked for his technical assistance, H. Beekhuizen for his participation in part of this study, and F. Kindt and W. Veenendaal (Department of Image-processing and Design) for their photographic services rendered. This research was supported by the Foundation for Fundamental Biological Re- search (BION), which is subsidized by the Netherlands Organiza- tion for the Advancement of Pure Research (ZWO).

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Received August 13, 1985 Accepted in revised form January 24, 1986