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Development 111, 829-844 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 829 Differential expression of two cadherins in Xenopus laevis B. ANGRES1, A. H. J. MULLER1, J. KELLERMANN2 and P. HAUSEN 1 * { 2 Max Planck Institut fttr Entwicklungsbiologie, Abt. fllr Zellbiologie, D-7400 Tubingen, Federal Republic of Germany Max Planck Institut fiir Biochemie, Genzentrum, D-8033 Martinsried, Federal Republic of Germany * Author for correspondence Summary Using a cadherin fraction from Xenopus tissue culture cells as an immunogen, two monoclonal antibodies were obtained that allowed the characterization of two distinct cadherins in the Xenopus embryo. The two cadherins differ in molecular weight, in their time of appearance during development and in thenspatial pattern of expression. One of the antigens was identified as E-cadherin. It appears in the embryonic ectoderm during gastrulation when epidermal differentiation commences and it disappears from the neural plate area upon neural induction. The second antigen could not be allocated to any of the known cadherin subtypes and was termed U-cadherin. It is present in the egg and becomes deposited in newly formed inner cell membranes during cleavage, the outer apical membranes of the embryo remaining devoid of the cadherin throughout development. U-cadherin is found on membranes of all cells up to the late neurula stages. A conspicuous polarized expression of the antigen on the membranes of individual inner cells suggests its participation in the segregation of cell layers and organ anlagen. These findings are discussed in the context of current hypotheses on the role of cadherins in establishing the spatial structure of the embryo. Introduction the aggregation of cells into distinct groups is accomplished by this differential expression of cadherins (for review see Takeichi, 1987). Examples of this relationship between cadherin expression and cell sorting in early development in mouse and chicken are the separation of mesodermal cells from the epiblast (Thiery et al. 1984), the segregation of the neural tube from the presumptive epidermis (Thiery et al. 1984; Crossin et al. 1985; Hatta and Takeichi, 1986) and the release of the neural crest cells from the neurectoderm followed by their integration into various tissues of the developing embryo (Aoyama et al. 1985; Hatta et al. 1987). Most of our knowledge on the nature of cadherins and their expression in adult and embryonic tissues is derived from observations made on vertebrates other than amphibians although research on amphibian development has traditionally emphasized aspects of cell interactions during embryogenesis. Few observations on Xenopus cadherins have been reported. Nomura et al. (1986) demonstrated that a calciumdependent cell-cell adhesion system is operating in the early Xenopus embryo. The trypsin sensitivity of this system is typical for cadherins and the ability of E-cadherin-positive F9 cells to bind to blastomeres indicates the presence of cadherins on embryonic cells The various ways by which cells adhere to each other has long been a focus of attention in the discussion on embryonic cell interactions (Townes and Holtfreter, 1955). Cell-cell adhesion is known to be mediated by specific molecules, including the cadherins (for reviews see Takeichi, 1988; Edelman, 1988; Kemler etal. 1989). Cadherins represent a family of closely related transmembrane proteins that require calcium for stability and function. Prominent members of this family are E-cadherin (=uvomorulin), P-cadherin and N-cadherin identified in mouse and their homologues present in other vertebrates. The interest in the function of the cadherins in embryogenesis derives from their cell and tissue specificity. Cadherin-mediated cell-cell adhesion is accomplished by the homophilic interaction of the extracellular domains of these transmembrane proteins. The molecular interaction between cadherin subtypes is homotypic. When different cadherins are present on different cells within a population, differential adhesion and cell sorting is seen to occur (for review see Takeichi, 1990). A changing pattern of cadherin expression has been observed in the course of vertebrate embryogenesis. It has been proposed that Key words: Xenopus laevis, cadherin, cell adhesion, development, cell polarity. 830 B. Angres and others from blastula to neurula. Levi et al. (1987) studied cadherin expression in Xenopus embryos using an antibody directed against L-CAM, which is thought to be the chicken homologue of E-cadherin. A diffuse staining within the cytoplasm of all cells up to stage 20 was observed. No preferential staining of cell membranes was seen before stage 9, in later stages some staining of the cell borders was observed. Choi and Gumbiner (1989) prepared monoclonal antibodies against a putative Xenopus E-cadherin. This cadherin was detectable in the embryo at the onset of gastrulation and was localized predominantly on cell membranes of the ectoderm. Recently, Choi etal. (1990) and Herzberg et al. (1990) identified antigens reacting with antibodies directed against the intracellular domains of cadherins. The observations indicate the presence of cadherins distinct from E-cadherin in early stages of the embryo, but these molecules remain poorly defined. Detrick and coworkers (1990) reported on the appearance of N-cadherin in the neural plate of gastrulating embryos and suggested that this cadherin participates in the segregation process of the neural tube. In this communication, we report on the characterization of two different cadherins present in the early Xenopus embryo. One of them represents the Xenopus homologue of E-cadherin. Details of its expression pattern complement the earlier observations of Choi and Gumbiner (1989). It first appears during gastrulation in the ectoderm and shows a distribution pattern that supports the hypothesis of the generation of specific embryonic regions by differential cadherin expression as described above. The second cadherin (U-cadherin) is ubiquitously expressed in all cells of the embryo from the first cleavage up to the late neurula stages. This overall distribution has not been described for a cadherin before and indicates that this cadherin is present on all cells during cell layer formation and the segregation of the early organ anlagen. However, after organ segregation U-cadherin is localized on the border cells in a polarized fashion with the boundary of the organ anlage being devoid of cadherin. Cadherins may thus participate in the segregation processes in a manner more sophisticated than previously thought. Similarly, the polar character of the blastomeres produced by the first cleavages is indicated by the restriction of U-cadherin to the newly formed, i.e. basolateral, membranes. Thus, the polarized distribution of cadherins typical for epitheha is established from the first cleavage onwards and is maintained in the outer cell layer of the embryo during further development. The implications of the observations for diverse processes of embryogenesis are discussed. Materials and methods Embryos Embryos were obtained as described by Fey and Hausen (1990) and staged according to Nieuwkoop and Faber (1967). Cell lines and tunicamycin treatment The A6 Xenopus kidney epithelia cell line, purchased from the American Type Culture Collection (Maryland, USA) was maintained in 85% RPMI 1640 (GIBCO) containing 10% fetal calf serum (GIBCO) at 26°C in a 5 % CO2 atmosphere. For tunicamycin treatment, a half confluent A6 monolayer culture was incubated with lO/igmT1 tunicamycin (Sigma) in culture medium for 22 h and used for the preparation of cell lysates as described below. SDS-polyacrylamide gel electrophoresis and immunoblotting SDS-PAGE was carried out according to Laemmli (1970). Silver staining of gels was performed as described by Morrissey (1981). For immunoblotting, proteins were electrophoretically transferred to nitrocellulose membranes. Blots were blocked in 5% (or 10%) low-fat milk in PBS and immunostained as described by Fey and Hausen (1990) with primary antibody (ascites, diluted 1:500-1:5000) and with secondary antibody (alkaline phosphatase or peroxidaseconjugated goat anti-rabbit IgG; Dianova). Antibody binding was visualized with O^mgml" 1 bromo-chloro-indolylphosphate and 0.33 mgml" 1 nitroblue tetrazolium (Sigma) in 0.1 M Na2CO3, pH10.2 or with the ECL western blot detection system (Arnersham). Preparation of extracts from cells and embryos for immunoblotting For preparation of cell lysates, one 10cm Petri dish of a confluent grown monolayer of A6 cells or stationary grown fibroblasts was washed three times with PBS, scraped off the Petri dish and heated to 95 °C in 100 /il SDS-gel loading buffer for 15min. Cell debris was sedimented by centrifugation and the supernatant was sonicated, to fragment residual DNA. Extracts from embryos were prepared by homogenization at 4°C in extraction buffer (10 /A per embryo; 10 mM Tris-HCl, pH7.4, 1.5mM CaCl2, 0.6mM MgCl2, 2% NP40) containing the following protease inhibitors: lmM phenylmethylsulfonylfluoride, lmM N-ethylmaleimid, 0.02mM leupeptin, 0.028 mM pepstatin, lO^gml"1 aprotinin, 1 mM iodoacetamid, 1 mM benzamidin. Homogenates were centrifuged at 5000revsmin"1 and 4°C for 3min (Heraeus, minifuge). The supernatant was extracted with an equal volume of 1,1,2 trichlorfluorethane and centrifuged in a Beckman SW65 rotor at 100 000 £ and 4°C for 30min. Purification of the extracellular domain of Xenopus E-cadherin Confluent monolayers of A6 cells were washed three times with digestion buffer (20mM Hepes, pH7.4, 5mM KG, 150 mM NaCl) supplemented with 1.5 mM CaCl2 and 0.6 mM MgCl2 and scraped off the Petri dishes (10 cm in diameter) in 4 ml digestion buffer containing 0.01% trypsin and either 1.5mM CaCl2 and 0.6mM MgCl2 or lmM EDTA. Cells were incubated on a rotary shaker at 80revsmin~' and 37 °C for 45min. The trypsin digestion was stopped by addition of 150 U of Kallikrein inhibitor (Trasylol; Bayer) and cells were pelleted by centrifugation (Heraeus, minifuge) for 5min at 2000 revs min"1 and 4°C. To remove residual cell debris the supernatant was centrifuged in a SW 34 rotor (Sorvall) for 20min at 9000 revs min"1 and 4°C. Supernatants of Ca2+-trypsin digestions were loaded on a 4ml lentil lectin-Sepharose column. Adsorbed material was eluted with 200 mM o^methyl mannoside. The eluate was concentrated and washed with PBS in a centricon 30 microconcentrator (Amicon). Two cadherins in Xenopus laevis Microsequencing 80 ng of the lentil-lectin-purified material was applied to a 10% SDS-PAGE according to Laemmli et al. (1970). The separated proteins were electroblotted on a siliconized glass fiber sheet (Glassybond, Biometra) and the 9Qx\&MT protein band was excised and directly placed into a gas-phase sequencer (477 A, Applied Biosystems). Establishment of hybridoma cell lines; monoclonal antibodies and control IgG Protein from 32 ml of Ca2+ trypsin digest was precipitated with trichloracetic acid and electrophoresed according to Laemmli (1970). 90xl(fiMT protein bands were excised after visualization with copper staining (Lee etal. 1987), destained, equilibrated with PBS and used for the immunization of mice. Animals with positive titers were boosted once more intravenously with 5/ig of lentil-lectin-purified protein in 200^1 PBS. Mouse hybridoma cell lines were established as described by Kearney et al. (1979) and Galfre' and Milstein (1981). Inert control IgG was produced by the myeloma cell line P3K (Horibata and Harris, 1970). Established cell lines were used to induce ascites growth in mice. The IgG fraction from ascites fluid was obtained by affinity chromatography on CM Affi-Gel Blue (Biorad) as a first purification step and subsequent ion exchange chromatography on a Mono Q column (Pharmacia). Immunohistology Organs of adult frogs were frozen in liquid nitrogen; stage 47 tadpoles were fixed in 2 % TCA for 3h, embedded in Tissue Tek (Miles Laboratories, Naperville) and frozen in liquid nitrogen. 10pun frozen sections were cut with a Reichert Jung microtome, mounted on gelatine-coated coverslips and fixed in acetone at —20°C overnight. Sections were dried shortly at —20°C and rehydrated in PBS at room temperature. Staining was performed with a 1:500 dilution of ascites fluid. Monoclonal antibody binding was detected with goat F(ab)2 anti-mouse IgG-FITC reagent (Dianova). Stained specimens were mounted in Mowiol (Hoechst), examined with an epifluorescence Axioplan Zeiss microscope. Whole embryos of different stages and isolated dorsal blastopore lip regions (stage 10i) were fixed in 20% dimethylsulfoxid, 80% methanol (Dent etal. 1989) overnight at -20°C. After several washes in PBS during 1 h at room temperature, the vitelline membrane of whole embryos was removed (stage 2-9) or embryos were cut into halves (stage 13-21) to improve penetration of the antibody. Specimens were incubated with antibody (diluted in 20 % rabbit serum in PBS) overnight at 4°C on a rocking platform followed by washes in PBS during the day at room temperature by changing the buffer several times. The incubation with mAb 6D5 or mAb 10H3 or inert P3 IgG (all ascites 1:500) was followed by the incubation with FITC-conjugated goat antimouse F(ab)2 as the secondary antibody (Dianova) and a FITC-conjugated rabbit anti-goat F(ab)2 antibody specific for the F(ab)2 fragment (Dianova) as the tertiary antibody. Embryos were incubated in 2 % paraformaldehyde in PBS for l h at room temperature and washed twice with PBS for lOmin each. Specimens were embedded in 2% agar and dehydrated in DMP (3,2 dimethoxypropane, Muller and Jacks, 1975) twice for 30min each. They were embedded in glycolmethacrylate (RWL, Histotechnologie, Bruckmiihl/ Vagen, FRG) as described by the supplier. 4 fan thick sections were cut on a Reichert Jung microtome and mounted in Mowiol (Hoechst, FRG). 831 Aggregation assays A6 cells were grown in 85 % Leibovitz medium containing 10 % fetal calf serum at 26 °C in microtiter plates coated with gelatine until they reached confluency. Cells were washed once with 85 % Leibovitz medium to remove loose cells and incubated in 85 % PBS. After they lost intercellular contacts but still remained attached to the bottom of the plate, cells were incubated in 85 % Leibovitz medium at room temperature supplemented either with purified IgG of mAb 6D5 or mAb 10H3 or with P3 IgG at a concentration of 10/igml"1 each. The reaggregation was observed with an inverted microscope. To test the aggregation of early cleavage blastomeres fertilized eggs were demembranated and maintained in Ca2+and Mg^-free Modified Barth's Solution (MBS-H; 88 mM NaCl, lmM KC1, 2.4mMNaHCO3, 0.82mM MgSO4, 0.33mM Ca(NO3)2, 0.41mM CaCl2, 10mM Hepes (+NaOH), 1% streptomycin, 1% penicillin, pH7.4) in a 24-well tissueculture plate coated with BSA ( 1 % solution, 1-2 h). When eggs had produced eight dissociated blastomeres, Ca2+ and Mg2"1" was added to the final concentration of 0.74 mM and 0.82 min, respectively. Purified IgG of mAb 6D5 or 10H3 or P3 as a control were added to a final concentration of lO^gml"1. The aggregation was followed with a stereomicroscope and photographs were taken after 2h (128-256 cells). Inner animal cap cells from stage 9 blastulae were isolated and incubated in Ca2+-Mg2+-free MBS-H containing lmM EDTA for l h on Petri dishes coated with 1% agarose. Dissociated cells were briefly washed in MBS-H and cells of one embryo were incubated on 1 % agarose in microtiter wells either in MBS-H supplemented with purified IgG of mAb 6D5, mAb 10H3 or P3 as a control at a concentration of 10/igml"1. A further control culture was maintained in the Ca2?/Mg2+-free condition. Results Preparation and characterization of antibodies against Xenopus cadherins A property common to all cadherins is the release of their extracellular domain from cells upon treatment with trypsin (Hyafil et al. 1980). The released fragment is stable to further trypsin action only in the presence of calcium ions: omission of calcium from the digest leads to the degradation of the cadherin fragment. A prominent protein fragment of SHJXlO3^,. was released from Xenopus A6 cells by trypsin incubation in the presence of calcium (Fig. lb). This fragment was not found in trypsin digests obtained in the absence of calcium (Fig. la). After loading the calcium-trypsin digest onto a lentil lectin column, the 90xlCrM r fragment was absorbed (Fig. lc) and could be released by cr-methyl mannoside as a rather homogeneous fraction (Fig. Id). Material from the lentil lectin column was further purified by preparative SDS-PAGE and applied to a gas-phase sequencer. The sequence of a segment of 21 amino acids at the N terminus was determined. Comparing this sequence with the corresponding sequences of several cadherins from other species reveals a high degree of homology (Fig. 2). These data indicate that the extracellular domain of one cadherin predominates in the isolate, though they 832 B. Angres and others a b e d e <90 — f g n ^90 m t Fig. 1. Characterization of the antigens from A6 cells recognized by mAbs 6D5 and 10H3. (a-d) Isolation of a 90xl(fi MT protein fraction from supernatants of A6 cells after trypsin treatment. Supernatants from trypsin-digested A6 cells were used for lentil lectin affinity chromatography as described under Materials and methods. Lane a: supernatant of A6 cells treated with trypsin in the absence of Ca (500^1 per lane). Lane b: supernatant from A6 cells treated with trypsin in the presence of Ca (500^1 per lane). Lane c: wash through of the lentil lectin chromatography (500^1 per lane). Lane d: eluate from the lentil lectin column (300 ^il per lane which corresponds 10% of the eluate). The arrowhead indicates the 90X103 Mr protein fraction, (e-h) Identification of the 90X103 MT protein fraction by mAbs 6D5 and 10H3 on immunoblots. Supernatants from A6 cells treated with trypsin in the presence of Ca2+ (lanes e and g) or in the absence of Ca2+ (lanes f and h) were immunoblotted with mAb 6D5 (lanes e and f) or mAb 10H3 (lanes g and h). (i-m) Determination of a protein moiety as epitopes for mAbs 6D5 and 10H3. Cell lysates of untreated A6 cells (lanes k and m) or tunicamycin-pretreated A6 cells (10/igmP 1 ) were immunoblotted with mAb 6D5 (lanes i and k) or mAb 10H3 (lanes 1 and m). Approximately 100 ng of protein was loaded on each lane. Arrowheads indicate the two main protein bands recognized by the two antibodies. Bars indicate relative molecular masses from top to bottom as follows: 200, 116, 97, 68, 43 (xlO3). do not establish that we have purified one protein to homogeneity. Mice were immunized with SDS-PAGE-purified ^ x l t ^ M f fraction and hybridoma lines producing antibodies were established. Two monoclonal antibodies, 6D5 and 10H3, were used for the further work. Both antibodies recognized exclusively the 90X103 Mr material released from A6 cells by calciumtrypsin treatment on immunoblots (Fig. le,g). No antigen was detected in the corresponding preparations obtained in the absence of calcium (Fig. lf,h). When whole-cell lysates were electrophoresed and immunoblotted, both antibodies bound to a 140x 103 Mr protein band that probably represents the intact, uncleaved cadherin molecule (Fig. lk,m). Antibody 10H3 also reacted with a band of 155xl(rM r , which presumably represents a precursor molecule, as well as with some degradation products of lower relative molecular mass. A6 cells were pretreated with tunicamycin to determine whether the antibodies recognize oligosaccharide epitopes. In addition to the intact cadherin, both antibodies recognize components of approximately 120 xlO 3 ^,. in the lysates from tunicamycintreated cells (Fig. li,l). These components presumably represent N-linked oligosaccharide-deficient cadherins that form in the presence of the antibiotic. Thus, it is likely that the antibodies are directed against protein epitopes on the cadherins. We investigated whether the two antibodies, 6D5 and 10H3, interfere with cell-cell adhesion using the following assay. Confluent monolayers of A6 cells were incubated in calcium-free buffer until the cells were released from intercellular contacts but still adhered to the bottom of the culture dish (Fig. 3A). Calcium was restored by replacing the buffer by normal culture medium, supplemented either with mAb 10H3, mAb 6D5, or with control P3 IgG. Within 2 h, an epitheliumlike monolayer reformed in the presence of P3 IgG (Fig. 3B). In the presence of either antibody, the cells assumed a fibroblast-like morphology and did not form the close cell contacts of an epithelium (Fig. 3C, D). These results indicate the usefulness of the two antibodies for functional studies of cell-cell adhesion. Different cadherins exhibit characteristic tissue distribution. In adult Xenopus tissues, the two antibodies stained the basolateral membrane domains of epithelial cells preferably at the apical junctional complexes in proximal and distal kidney tubules, and in lung Two cadherins in Xenopus laevis 90kD 833 p.f. 75 79 60 Q E L 65 Q R L 65 Q E L 65 Q R L 65 Q E L 65 Fig. 2. Comparison of the 21 N-terminal amino acids from a microsequence analysis of the 90X103 MT protein fraction and the corresponding amino acid sequences of cadherins in different species. Residues identical with those of the 90x10* MT protein fraction are enclosed in boxes. Numbers on the right indicate % identity of the obtained 90X103 Mt protein fraction with the listed cadherins. The N terminus is marked by an arrowhead. X=not identified amino acid residue; M, mouse; H, human; C, chicken; X, Xenopus; cad, cadherin; p.f., protein fraction. Sequences from top to bottom are reported by Nagafuchi et al. 1987 and Ringwald et al. 1987; Wheelock et al. 1987; Gallin et al. 1987; Miyatami et al. 1989; Nose et al. 1987; Hatta et al. 1988; Shimoyama et al. 1989; Detrick et al. 1990. epithelia (not shown). Sections of the head region of a stage 47 tadpole showed an intense staining at the basolateral cell membranes of the epidermis (Fig. 4A,B). No staining was seen in the brain tissue. This observation suggests that the antibodies do not react with N-cadherin, which typically occurs in brain tissue (Hatta et al. 1985). The staining of liver sections with antibody 10H3 appears as pairs of parallel lines in the parenchyma (Fig. 4D). These lines presumably indicate boundaries of the bile canaliculi since the same staining pattern was observed by using antibodies against cell junctional molecules (Tsukita and Tsukita, 1989; Stevenson et al. 1986). Staining of these structures was not observed with antibody 6D5 (Fig. 4E). In heart tissue sections, antibody 6D5 staining appeared as lines perpendicular to the length of cardiac muscle cells (Fig. 4H, arrows) and as a repetitive pattern along the side of muscle cells (Fig. 4H, arrows). We presume that these patterns indicate staining of intercalated discs and the costameres of the heart muscle cells, structures that are believed to mediate firm cohesion between the successive cellular units in the myocardium (intercalated discs) and the physical contact to the underlying myofibrils (costameres) (Tsukita and Tsukita, 1989; Pardo et al. 1983). No such structures were stained with mAb 10H3 (Fig. 4G). Antibodies 10H3 and 6D5 recognize different cadherins with different expression schedules in the embryo The finding that both antibodies stain basolateral membrane domains and the junctional complexes in Fig. 3. Antibody inhibition of the reaggregation of dissociated A6 cells. Cells of an A6 monolayer culture were dissociated in Ca2+-free buffer (A) and incubated for 2h in culture medium supplemented with either control IgG P3 (B), purified mAb 10H3 (C) or mAb 6D5 (D) in a concentration of 10/xgml"1 each. Bar, 50[im. epithelia and specialized adhesive structures in nonepithelial tissues agrees with their specificity for cadherins. The observation that specific structures in heart and liver are each stained by only one of the two antibodies provided the initial indication that the two antibodies recognize different cadherins. This latter notion was verified when the antibodies were applied in studies of the development of the Xenopus embryo. As in A6 cell lysates, antibody 10H3 recognized a molecule of 140xl0 3 M r in the embryo extracts (Fig. 5A, a and b). In contrast, antibody 6D5 recognized a protein of about 125 x l O 3 ^ in the embryo extract, which can easily be distinguished from the corresponding A6 cell antigen of 140x10? MT (Fig. 5A, c and d). Thus, the two antibodies, 10H3 and 6D5, recognize different cadherin subtypes in the embryo. The time of appearance of both cadherins during early development was determined by western blot analysis of extracts obtained from embryos at different stages ranging from the fertilized egg (stage 1) to the late neurula (stage 20). The result is shown in Fig. 5B and C. The cadherin recognized by mAb 10H3 was first found in the embryo at gastrulation thereafter increasing in amount. In contrast, the antigen of mAb 6D5 was already present at stage 1 and increased in amount up to the gastrula stages (Fig. 5C). This differential timing of the expression of the two antigens further strengthens the notion that we have identified two different cadherin molecules. On the basis of its staining pattern in different tissues, the molecular weight data and the time of its expression during embryogenesis, we conclude that antibody 10H3 834 B. Angres and others Fig. 4. Immunostainings of Xenopus tissues. Cryostat sections of the head region of a stage 47 tadpole (A-C), liver (D-F) and heart (G-I) were immunostained with mAb 10H3 (A,D,G), mAb 6D5 (B,E,H) or inert control IgG (C,F,I). Arrows and arrowheads inert control IgG (C,F,I). Arrows and arrowheads in H indicate the intercalated discs and the costameres in heart tissue, respectively. Bars, 30//m. recognizes the Xenopus homologue of E-cadherin, which has been characterized by Choi and Gumbiner (1989) (for further details see Discussion). Antibody 6D5 recognizes a cadherin antigen that has not yet been characterized in Xenopus and which cannot currently be allocated to any of the known subtypes of cadherins (see discussion). We will provisionally use the term U-cadherin for this component in further discussion. U-cadherin participates in cell-cell adhesion from early cleavage onwards The early presence of U-cadherin in the embryo suggests that it might mediate cell-cell adhesion during the cleavage stages. E-cadherin should not be involved since it is not present at this time. Functional assays support these assumptions. Demembranated fertilized eggs were placed in calcium- and magnesium-free buffer solution to avoid cell-cell adhesion between the Two cadherins in Xenopus laevis Fig. 5. Identification of two different cadherins in embryos by mAbs 10H3 and 6D5. Protein extracts of embryos of different stages were prepared as described in Materials and methods. The equivalent of 5 embryos per lane was used for electrophoresis and immunoblotting. (A) Comparison of molecular masses of cadherins recognized by mAbs 10H3 and 6D5 in protein extracts of stage 20 embryos. A6 cell lysates (100/ig/lane, lanes a and d) and the protein extract of stage 20 embryos (lanes b and c) were immunoblotted with mAb 10H3 (lanes a and b) or mAb 6D5 (lanes c and d). (B) and (C) Western blot analysis of the antigens recognized by mAbs 10H3 (B) and 6D5 (C) during development. Stages from a-g: 1, 3, 8 10, 12, 14, 20. Numbers indicate relative molecular masses xlO3. a b e d -200 -116 -97 -66 -43 B a b c d e f g -200 -116 -97 -66 -43 C a b c d e 835 f 9 -200 -116 -97 -66 -43 blastomeres during the first cleavages. After the third cleavage division, the buffer was reconstituted with the divalent cations in two of three samples. One of these samples received antibody 6D5, the other antibody 10H3. Cell division proceeded unimpaired in all three samples. After an additional five division cycles, the blastomeres in the buffer free of divalent cations formed a loose assembly. The cells were round and formed only loose point contacts (Fig. 6A). In the sample containing divalent cations plus antibody 10H3, the blastomeres were in the process of aggregation. The membranes of neighbouring cells formed broad contacts with cell shape adapting to these contacts (Fig. 6B). The same pattern of aggregation was observed when blastomeres were incubated with inert P3 control IgG (not shown). In the sample containing antibody 6D5, the appearance of the cell assembly resembled that of the control deficient in divalent cations, but cell contacts seemed to be tighter (Fig. 6C). Thus, an effect of the antibody on cell aggregation was clearly visible, but the cell contacts were not completely obliterated. These results demonstrate that U-cadherin is functionally active during early cleavage stages whereas cell aggregation is not affected by the presence of the antibody against E-cadherin. The differential effect of the antibodies on cell aggregation was even more pronounced in a functional assay using disaggregated cells from the inner layer of the blastocoel roof of stage 9 embryos. The assay was performed in a way similar to that described for the early blastomeres. 5h after reconstituting the medium with divalent cations in samples containing antibody 10H3, these cells formed tight aggregates (Fig. 6E). In samples free of divalent cations, no adhesion between the cells was observed (Fig. 6D). In samples that had received antibody 6D5, cell-cell adhesion was severely impaired (Fig. 6F). The cells were competent to associate but they remained spherical and did not form compact aggregates as they did in the presence of antibody 10H3. The same experiment was performed with inner vegetal cells, yielding the same results (not shown). To test whether the residual adhesion in the presence of antibody 6D5 was due to E-cadherin, the assay was performed in the presence of both antibodies. The result did not differ from that obtained by incubating the cells with 6D5 alone (not shown). The differential effect of the two antibodies on cell adhesion supports the notion deduced from the western blot analysis that they recognize different adhesion molecules in the embryo. The residual aggregation that 836 B. Angres and others Fig. 6.' Aggregation assays of blastomeres of the early embryo and inner cells of the animal cap of late blastulae. (A-C) Fertilized eggs were demembranated and incubated in Ca2+-free buffer until the 8-cell stage was reached. The incubation was continued in Ca2+-free buffer (A) or in Ca2+-containing buffer supplemented with 10/igmF 1 of purified mAb 10H3 (B) or mAb 6D5 (C). Photographs were taken 4h post-fertilization. (D-F) Inner cells of the animal half of a stage 9 blastula were isolated and dissociated in Ca2+-free buffer containing lmin EDTA. Cells were further incubated either in Ca2+-free buffer (D) or in Ca2+-containing buffer supplemented with purified mAb 10H3 (E) or mAb 6D5 (F) in a concentration of lO^gml" 1 each. Photographs were taken after 5h of incubation. By this time control embryos had reached stage 13. Bars, 500/an. occurs in the presence of mAb 6D5 remains to be explained. It is conceivable that, apart from the adhesion mediated by U-cadherin, further calciumdependent cell-cell adhesion systems are operating in the early developmental stages. The expression of E-cadherin is restricted to the ectoderm and disappears from cells of the neural plate. Whole embryos at different stages were fixed and immunostained, embedded in methacrylate and sectioned (see Materials and methods). With this method, E-cadherin was first detected in the outer epithelial cells at the original animal pole of stage 12 embryos (not shown). In sagittal sections of stage 12.5 embryos (late gastmla), the staining was more pronounced (Fig. 7B). It was restricted to the ectodermal layer and extended equally from the original animal region to the dorsal side (including the neural plate) and to the ventral side of the embryo (Fig. 7B). The signal intensity was highest at the original animal region and decreased gradually towards the vegetal pole, such that no staining was detected at the dorsal and ventral blastopore lips. In stage 14 embryos (early neurula), the staining increased in intensity as compared to stage 12.5 and was evenly distributed in the presumptive epidermis (Fig. 7E). Cells of the ectoderm forming the neural plate were almost devoid of the antigen. In stage 20 embryos (late neurula), cells of the closed neural tube Two cadherins in Xenopus laevis 837 np Fig. 7. Distribution of E-cadherin in the embryo during early development. Embryos were immunostained with mAb 10H3 embedded in glycolmethacrylate and sectioned. A schematic drawing (A) indicates the cell layers seen in the sagittal section of an embryo at stage 12.5 (B). The arrow in A indicates the original animal pole region. C represents a magnification of the original animal pole region in B. (D) A schematic drawing indicates the cell layers seen in the transverse section of an embryo stage 14 (E). (F) Transverse section of an embryo stage 20. np, neural plate; pe, presumptive epidermis; bl, blastocoel; ae, archenteron, dbl, dorsal blastopore lip; vbl, ventral blastopore lip; be, bottle cells; no, notochord; pm, paraxial mesoderm; ar, archenteron roof; nt, neural tube;. Bars represent in B and E 200/an, in C and F 50/an. 838 B. Angres and others were completely unstained while the cells of the epidermis strongly expressed E-cadherin (Fig. 7F). At all stages, the outer, epithelial, layer of the ectoderm was more intensely stained than the inner, sensorial, layer. Membrane staining in the epithelial layer was restricted to the basolateral membrane domains, the apical membranes at the embryonic surface were devoid of the antigen. Membranes of the sensorial cells were uniformly decorated by the antibody. In our interpretations, we have focussed only on membrane staining although additional intracellular staining has been observed. This intracellular staining, which occurs preferentially in the nuclear area, is difficult to interpret since a signal in this area was also produced in controls in which the cadherin-specific antibody was omitted from the staining procedure. (Fig. 9B and 10D). However, the observation that the intensity of the intracellular staining is greatly enhanced in regions that also exhibit a clear membrane staining might indicate the presence of some intracellular antigen. • • * U-cadherin stains all cells from first cleavage to late neurula Although the western blot analysis' revealed the expression of U-cadherin in the fertilized egg, no staining of the egg membrane was observed (not shown). This indicates a scattered distribution within the egg cytoplasm of this protein before the first cleavage division. When the first cleavage division occurs, the cadherin appears only on the newly formed membranes (Fig. 8A, arrows). The original outer plasma membrane marked by the underlying pigment granules (Fig. 8B) remains unstained. This absence of antigen on the apical membrane of the outer embryonic cells is maintained throughout development. Internally, the antibody stains all cells during the cleavage stages; no particular cell groups are marked by the antibody, nor do specific membrane regions lack the antigen. In Fig. 9, the distribution of the cadherin on cell membranes of the dorsal blastopore lip region of a stage 10i embryo is shown. Cell membranes are decorated by the antibody in all three germ layers in equal intensity. An intensified fluorescent signal at the apico-lateral cell borders of the bottle cells at the blastopore indicates a concentration of the antigen at this site by the apical constriction (arrowhead). Fig. 10 depicts the antigen distribution in the dorsal region of the midgastrula, of the neural groove stage and of the closed neural tube stage, in transverse sections. Fig. 10A demonstrates the polar expression of the antigen in the epithelial layers of the ectoderm and the archenteron roof. Only the basolateral membrane domains are stained, the apical membranes being devoid of the antigen. In favourable instances, the antigen is seen concentrated in the junctional complexes of these epithelia (arrows). Polar distribution of the antigen is maintained in the neurectoderm when the neural tube is invaginating (Fig. 10B). The inner cells B Fig. 8. Localization of U-cadherin to the newly inserted cell membranes in the first cleavage stage. Embryos in the first cleavage stage were immunostained with mAb 6D5, embedded in glycolmethacrylate and sectioned. (A) Immunostaining. (B) Same section in phase contrast. Arrows indicate the staining of newly formed membranes in the first cleavage (A). Bar, 50 jan. initially display a more even distribution of U-cadherin on their membranes. Later, when the segregation of the anlagen proceeds, a polar expression becomes obvious on internal cells as well (Fig. 10B,C, arrows). In the anlagen of notochord, somites and neural tube, the antigen is expressed on membranes that contact their homotypic neighbours within the organ anlage, but is absent from the membrane domains that delimit the anlage from the surrounding cells. Two cadhehns in Xenopus laevis 839 Fig. 9. Distribution of U-cadherin in the dorsal blastopore lip region. Pieces of embryos stage 10.5 containing the dorsal blastopore lip region were stained with mAb 6D5 (A) or control IgG P3 (B), embedded in glycolmethacrylate and sectioned. Arrowhead indicates apically constricted bottle cells. Arrows point in the direction of the animal pole, en, endoderm; me, mesoderm; ec, ectoderm. Bar, 100 ^m. Discussion Using a cadherin fraction from Xenopus A6 cells as immunogen, two mouse monoclonal antibodies, 10H3 and 6D5, were prepared that recognize different types of cadherin in Xenopus. Antibody specificity That the antibodies are specific for cadherins is based on the following observations: both antibodies recognize a calcium-protected fragment that is released by trypsin from the surface of whole cells; their antigens display the typical basolateral distribution of cadherins on epithelial cells; the antigens are also found on specialized adhesive structures of non epithelial cells. Further, both antibodies interfere with the calciumdependent cell-cell adhesion process in tissue culture. Several findings provide evidence that each of the two antibodies is directed against a different cadherin molecule. The two antigens are of different molecular weight in Xenopus embryos and the staining patterns of the two antibodies in embryos were strikingly different. Further, specialized adhesive structures found in adult heart and liver were stained only by one antibody and not by the other. Neither of the two antibodies is directed against an N-linked oligosaccharide epitope of the cadherins. The difference in antibody specificity does not, therefore, reflect a difference in the N-linked oligosaccharides of the cadherins. Interestingly, the two antibodies recognize antigens of different molecular weight in the embryo but both react only with a single antigen band on western blots of A6 cell lysates. The latter seems to be a true crossreaction as the antigen precipitated from A6 cell lysates with mAb 6D5 does react with mAb 10H3 on blots and vice versa (data not shown). It seems that the A6 cell antigen shares epitopes which are located on different molecules in the embryo. This question requires further investigations and may be solved when sequence data are at hand. Tissue distnbution of the antigens One criterium that defines different subclasses of cadherins is their pattern of occurrence in various tissues. In the attempt to allocate the antigens of mAb 10H3 and mAb 6D5 to known subclasses, their pattern of occurrence in Xenopus was compared to that of cadherins in mouse and chicken. As evident in Table 1, the expression pattern of 840 B. Angres and others Fig. 10. Distribution of U-cadherin in neurula stages. Anterior halves of embryos stage 13 (A) and dorsal halves of embryos stage 17 (B) and stage 21 (C and D) were stained with mAb 6D5 (A, B and C) or control IgG P3 (D). The tissues were embedded in glycolmethacrylate and transverse sections were cut through the middle of the dorsal regions. Arrows in B and C indicate cell membranes devoid of staining, np, neural plate; no, notochord; pm, paraxial mesoderm; ar, archenteron roof; af, archenteron floor; so, somite; nt, neural tube. Bar, 30/on. Two cadherins in Xenopus laevis 841 Table 1. Comparative distribution profile of different cadherins in adult tissues of Xenopus, mouse and chicken Xenopus Mouse Chicken Tissues E-cad. U-cad. E-cad.1 N-cad.1 P-cad.2 L-CAM3 N-cad.4 Skin Kidney Lung Liver Heart Brain + + + + - + + + — + — + +2 + + - n.r. — — + + + (+) (+) (+)* - + + + + — — + + n.r.=not reported; (+)=transiently expressed; *=expression in the epimyocardium; •Hatta et al. 1985; 2Nose and Takeichi, 1986; Thiery et al. 1984; 4Hatta and Takeichi, 1986. 3 antigen 10H3 in adult tissues matches that of mouse E-cadherin and its homologue L-CAM of chicken. Antigen 10H3 is distinguished from N-cadherin by its absence from brain and from P-cadherin by its presence in adult Xenopus kidney and lung. P-cadherin has also been reported to be present in mouse embryonic endoderm and mesoderm (Nose and Takeichi, 1986), whereas antigen 10H3 is confined to the ectodermal germ layer in the Xenopus embryo. Similar to the case for E-cadherin in mouse and chicken embryos, antigen 10H3 disappears from the neural plate of the Xenopus embryo while N-cadherin begins to be expressed in this location (Thiery et al. 1984; Nose and Takeichi, 1986; Detrick et al. 1990). Our data on antigen 10H3 with respect to molecular weight, timing of expression in the embryo and restriction to the embryonic ectoderm, agree with those reported for Xenopus E-cadherin (Choi and Gumbiner, 1989). For these reasons, we classify the antigen of mAb 10H3 as E-cadherin. The tissue distribution of the cadherin recognized by mAb 6D5 does not allow a clear allocation to any of the known types of cadherins (Table 1). It is distinguished from E-cadherin by the observations reported here. Its absence from the late tadpole brain tissue makes its classification as an N-cadherin, unlikely. Mouse P-cadherin is only transiently expressed in kidney and lung, while antigen 6D5 is present in these tissues in the adult frog. However, as nothing is known about the expression pattern of P-cadherin in Xenopus, the classification of this antigen remains unclear. A crossreaction of antibody 6D5 with further still unknown Xenopus cadherins is possible. For these reasons, we have provisionally chosen for the antigen of antibody 6D5 the term U-cadherin (ubiquitous in the early embryo). A polyclonal antiserum was made against a synthetic peptide derived from a highly conserved region of the cytoplasmic domain of cadherins (Choi et al. 1990). U-cadherin may be related to an antigen recognized by this antibody. This antigen was termed CLP (cadherinlike protein). It is present early in the embryo and its relative molecular mass agrees with that of U-cadherin. Similarly, Herzberg et al. (1990) described an antigen that is recognized by an antibody directed against the intracellular domain of mouse uvomorulin. This antigen occurs early in the Xenopus embryo. In both reports, the antigens are not sufficiently characterized to allow for a full comparison with U-cadherin. The relationship of these different components may become clear when sequence data become available. Spatial distribution of the cadherins raises questions on embryonic patterning E-cadherin appears in the embryonic ectoderm during gastrulation at a point when other molecular markers indicate the onset of epidermal differentiation. All of these markers, including E-cadherin, exhibit the common feature that they are expressed in a polarized fashion within the ectoderm, the epithelial layer being more heavily loaded with the component than the sensorial layer. These epidermal markers seem to fall into two classes. (1) A class of antigens that is expressed only later than stage 12.5. These antigens are confined to the prospective epidermal region, the prospective neural tissue remaining devoid of the markers (antigen 2F7.C7 (Jones and Woodland, 1986); antigens XEPI-1,2 and 3 (Itoh et al. 1988); antigen Epi-I (Akers et al. 1986)). (2) The other class of antigens includes an epidermal cytokeratin gene that is expressed at pregastrula (stage 9) in the animal region of the embryo, including the prospective neural plate region. When neural induction commences in the dorsal ectoderm, expression of the keratin gene is inhibited (Jamrich et al. 1987). E-cadherin is a member of this second class. With the most sensitive methods, we have been able to detect E-cadherin as early as stage 10. In stage 12.5 the neural plate area is stained with antibody 10H3 as well as the prospective epidermis. Later, E-cadherin disappears from the neural plate and the neural tube. These data support the concept that neural induction deviates ectoderm from its progression towards epidermis into the neurogenic pathway at a time when epidermal differentiation is already well under way. One effect of neural induction is then seen as an inhibition of epidermal differentiation. As neural development progresses, this condition does not allow the expression of the 'late' epidermal markers. An additional similarity in the spatial expression of epidermal cytokeratin and E-cadherin is their graded distribution in the animal-vegetal direction in the early phases. In both cases, the expression is initially highest in the animal region and decreases as one moves 842 B. Angres and others towards the vegetal pole. This pattern appears for cytokeratin mRNA in the stage 10.5 embryo (Jamrich et al. 1987) and for the E-cadherin molecule at the stage 12.5 embryo. At slightly later stages (stage 12 and 13, respectively), all of the prospective epidermis expresses the markers equivalently. In a transient phase, a wave of epidermal differentiation seems to pass over the ectoderm from the animal region towards the vegetal pole. In the mouse embryo, E-cadherin is already present on the membrane of the egg (Vestweber et al. 1987) and becomes restricted to the basolateral membrane domains during compaction when epithelial cell polarity is induced by cadherin-mediated contacts (Shirayoshi et al. 1983; Johnson et al. 1986). In Xenopus, U-cadherin, and not E-cadherin, is expressed in the early stages of development. The deposition of U-cadherin is restricted to the newly formed inner membranes which represent the basolateral membrane domains. A similar distribution has been proposed for the Na+-K+-ATPase (Slack and Warner, 1973). These observations underline the notion that cell polarity is a conspicuous feature from the first cleavage division onwards. The absence of U-cadherin allows us to define the apical character of the outer membranes using this molecular membrane marker. This apical property is an early feature of the egg membrane and is inherited by all membrane domains in the embryo that derive from the egg plasma membrane, i.e. the apical domains of the outer epithelium and of the archenteron roof up to the late neurula stages. The non-adhesiveness of the ectodermal apical membrane domain has long been recognized (Holtfreter, 1943; Roberson et al. 1980). We show here that this feature may be explained by the lack of U-cadherin on these membrane domains. The question of how the apical character of the egg membrane arises during oogenesis is currently being investigated (Miiller et al. unpublished data). From egg to the beginning of gastrulation, the area occupied by apical membrane domains does not increase. During cleavage, there is no need for de novo formation of outer apical membranes. Restriction of membrane formation to the membranes in the embryo's interior may thus maintain the apical-basolateral polarity of the outer cells. The plasma membranes formed in the embryo internally are generated by the fusion of preformed Golgi-derived vesicles (Sanders and Singal, 1975). We anticipate that these vesicles are endowed with U-cadherin before fusion. At stage 6 of development, the outer cells of the embryo begin to form a functional epithelium with the cell apices being joined by fully developed tight junctions (Miiller, unpublished observation). E-cadherin-mediated contacts seem to be a prerequisite for the formation of these junctions in tissue culture (Gumbiner and Simons, 1986) and in early mouse development (Fleming et al. 1989). In Xenopus, Ucadherin would serve this function in the pregastrula embryo. The inhibitory effect of antibody 6D5 on blastomere aggregation shows that U-cadherin is a prominent mean by which the early blastomeres are held together within the embryo. Thus, a major function of U-cadherin in early development is to maintain the integrity of the embryo. Gastrulation is accomplished by the movements of sheets of cells with no gross intermingling of the cells occurring within the sheets. The presence of U-cadherin on cells of the gastrulating embryo may facilitate the directed morphogenetic movement. When a sheet of mesodermal cells moving in an explant across the blastocoel roof is exposed to antibody 6D5, the cells readily disperse and begin to move around in a nonoriented fashion (Winklbauer et al. unpublished data). A participation of U-cadherin in the proper execution of the gastrulation movements is inferred from this observation. Mechanical forces generated by the movements of cell sheets must be distributed within the gastrulating embryo. The interconnection of the cells by U-cadherin may serve this function. The presence of U-cadherin on the motile cells poses a problem at the same time. To a limited extent, the cells do change position within one sheet in particular during epiboly and convergent extension (Keller, 1986). Moreover, whole cell collectives harbouring U-cadherin slide past each other. It remains unclear how these neighbouring cells, which are interconnected by the homophilic interaction of the cadherin, retain their mutual mobility. The cells obviously must possess means to modulate cadherin function locally on their surfaces. During gastrulation individual regions of the embryo begin to detach from their neighbours as they segregate into organ anlagen. It has been postulated that these segregation processes are accomplished by the spatially restricted expression of different cadherins (for review see Takeichi, 1988). The ubiquitous expression of U-cadherin on all cells would preclude its participation in such mechanisms. However, details in the spatial localization of U-cadherin on individual cells reveal a feature that may give an aid in the segregation processes. On individual cells U-cadherin becomes deposited in a polar manner, such that the membranes that touch each other within the anlage carry the antigen, whereas the cadherin is absent from the membranes at the outer border of the segregating cell group. The established cell group might lose adhesiveness this way. Coordinated with this cell polarization ECM becomes deposited around the organ anlagen (Fey and Hausen, 1990) and integrin becomes specifically expressed on the cell membranes facing the newly formed matrix (Gawantka, unpublished observation). Organ segregation seemingly requires the coordinated interplay of several molecular systems, the details of which need to be elucidated. 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