Schneider S et al. (1993), Catenins in Xenopus embryogenesis and their rel...

XB-ART-22553

Development 1993 Jun 01;1182:629-40.

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Catenins in Xenopus embryogenesis and their relation to the cadherin-mediated cell-cell adhesion system.

Schneider S , Herrenknecht K , Butz S , Kemler R , Hausen P .

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In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against alpha- and beta-catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that alpha- and beta-catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the catenins are complexed to U-cadherin in the early phase of embryogenesis and to E-cadherin, when it appears during gastrulation. An excess of alpha-catenin occurs in free form in the early embryo, whereas all of the beta-catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of alpha-catenin was analysed by immunohistology. During cleavage alpha-catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of alpha-catenin. At later stages, the pattern of alpha-catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of E-cadherin in the gastrula ectoderm is accompanied by conspicuous depositions of alpha-catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry alpha-catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

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Species referenced: Xenopus
Genes referenced: cdh1 cdh3 ctnnb1 mcf2

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	Fig. 1. Cross-reaction of antibodies directed against mouse a-catenin and b-catenin with Xenopus catenins. (A) Extracts of five embryos each of stage 10 (lanes 1, 3, 5 and 7) and stage 17 (lanes 2, 4, 6 and 8) were electrophoresed. Blots were developed with anti-U-cadherin (lanes 1 and 2), anti-E-cadherin (lanes 3 and 4), anti-a-catenin (lanes 5 and 6) and anti-b-catenin (lanes 7 and 8). (B) Components precipitated by anti-U-cadherin from extracts of 50 stage 10 embryos were analysed by immunoblotting using anti-U-cadherin (lane 1), anti-a-catenin (lane 3) and anti-b-catenin (lane 5). (C) Components precipitated by anti-E-cadherin from extracts of 50 stage 17 embryos were analysed by immunoblotting using anti-E-cadherin (lane 1), anti-a-catenin (lane 3) and anti-b-catenin (lane 5). For controls in B and C (lanes 2, 4, and 6), mock precipitates using an inert IgG were used for electrophoresis. The strong signals in the lower part of B and C result from IgGs which were released into the samples from the precipitation beads and which bind the second antibodies. The position (bars) and size (Mr) of molecular weight markers are indicated.
	Fig. 2. Western blot analysis of a-catenin, b-catenin, E-cadherin and U-cadherin during early embryogenesis. Extracts of embryos of different stages (five embryos per lane) were analyzed by immunoblotting with mAb 10H3 for E-cadherin, mAb 6D5 for Ucadherin, a-M12K for a-catenin and a-P14L for b-catenin. The developmental stages are indicated.
	Fig. 3. Coprecipitation of a- and b-catenin with U-cadherin and E-cadherin from extracts of embryos of different stages. (A) Immunoprecipitates obtained with anti-U-cadherin from extracts of 50 embryos at different developmental stages were analysed by immunoblotting for U-cadherin, and a- catenin and b-catenin as before (see Fig. 2). (B) Immunoprecipitates obtained with anti-E-cadherin from extracts of 50 embryos of different developmental stages were analysed by immunoblotting for E-cadherin and a- catenin and b-catenin as before (see Fig. 2). The developmental stages are indicated. co, controls.
	Fig. 4. Catenins in extracts depleted of U-cadherin by sequential immunoprecipitation. Extracts of 50 stage 10 embryos were subjected to two consecutive immunoprecipitations with anti-Ucadherin. Aliquots of the original extract (A) and the supernatants of the first and the second immunoprecipitation (Bs and Cs respectively) and the corresponding precipitates (Bp and Cp) were analyzed for U-cadherin, a-catenin and b-catenin by immunoblotting as before (see Fig. 2). Ds and Dp are supernatant and precipitate of the control samples in which inert P3-IgG was applied for precipitation.
	Fig. 5. Separation of a-catenin from U-cadherin and b-catenin by sucrose gradient centrifugation. An extract of 20 stage 10 embryos was fractionated on a sucrose gradient (see Material and Methods) and the fractions (fraction numbers from bottom to top) were analyzed for U-cadherin, a-catenin and b-catenin by immunoblotting as before (see Fig. 2). The arrowheads indicate the peak distribution of protein standards of known S values (from left to right: 11.4 S, 7.4 S, 4.6 S).
	Fig. 6. Analysis of metabolically labelled proteins that coprecipitate with U-cadherin and a-catenin. 30 fertilized eggs were injected with [35S]methionine and cultured until stage 8 and 30 stage 11.5 gastrulae were injected with [35S]methionine into the blastocoel and cultured until stage 13. Aliquots of extracts were immunoprecipitated with anti-a-catenin and with anti-Ucadherin respectively. Electrophoretic patterns of radioactive proteins were visualized by fluorography. After fluorography, Ucadherin, a-catenin and b-catenin were identified by staining the blots with the corresponding antibodies. Their position is indicated by arrowheads. The unidentified coprecipitating protein atMr 88´103 might be g-catenin. In the control samples, an inert IgG was applied in the precipitation. (A) Extracts of labelled stage 8 embryos. (B) Extracts of labelled stage 13 embryos. Lanes 1: controls; lanes 2: precipitates obtained with anti-a-catenin; lanes 3: precipitates obtained with anti-U-cadherin.
	Fig. 7. Distribution of a-catenin in early cleavage stage embryos. Embryos were immunostained with a-M12K antibody, embedded in glycolmethacrylate and sectioned. (A) Animal pole region of a fertilized egg. Arrowheads indicate the plasma membrane. (B) Animal pole region of an eight cell stage. The internal plasma membrane is indicated by arrowheads. (C) Blastomeres near the animal pole of a stage 6 embryo. The lateral membrane was sectioned tangentially (arrowheads). (D) Cleft (arrowheads) between vegetal blastomeres of a stage 6 embryo. (E) Transversal section of a stage 6 embryo. Arrowheads indicate the cell membranes adjacent to the blastocoel. No staining was obtained in controls. an, animal pole region; bl, blastocoel; vg, vegetal pole region; Bars: 50 um.
	Fig. 8. Distribution of a-catenin in blastula and gastrula stages. (A) Ventral portion of a sagittal section through a stage 10 embryo. (B) The marginal region of a stage 9 blastula. Apical concentrations of a- catenin along the lateral membranes of the outer ectodermal blastomeres are indicated with arrowheads. (C) Dorsal blastoporal lip region of an early gastrula with apically constricted bottle cells (arrowhead). (D) Lateral blastopore lip region of a stage 12.5 embryo. Arrowheads indicate apical membranes of the cells that form the archenteron roof and floor. The unspecific staining of the yolk platelets is due to autofluorescence. an, animal region; bl, blastocoel; dbl, dorsal blastopore lip region; en, endoderm; ec, ectoderm; me, mesoderm; vg, vegetal pole region; yp, yolk plug. Same histological procedure as in Fig. 7. Bars: 50 um.
	Fig. 9. Distribution of a-catenin in neurulae. (A) Transversal section of a stage 14 embryo. (B) Section through the lateral region of a stage 14 embryo depicting the ectodermal, mesodermal and endodermal cell layers. (C) Ventral detail of a sagittal section through a late neurula stage 19 embryo. Arrowheads point to the apical membranes of the endoderm. (D) Embryos at stage 17 and (E) at stage 19 were cut through the central dorsal region transversally. Arrows indicate the regions where cell membranes are devoid of staining. ae, archenteron; ar, archenteron roof; bo, bottle cells; ec, ectoderm; en, endoderm; me, mesoderm; no, notochord; pm, paraxial mesoderm; so, somites. Same histological procedure as in Fig. 7. Bars: 50 um.
	Fig. 10. Distribution of a-catenin in the stage 27 tailbud embryo. (A) Transversal section showing staining patterns in different organ anlagen, particulary in the neural tube, pronephros anlage (arrows) and lateral plate mesoderm (arrowheads). (B, C, F, G and H) Details of transversal sections; (D,E) details of parasagittal sections. (B) Somite, arrows indicate the inhomogenuous staining pattern around the somite cells. (C) Cement gland, arrows point at a lateral plasma membrane of a columnar cell. (D) Eye vesicle. (E) Notochord, plasma membranes bordering the basal lamina are indicated by arrows. (F) Neural crest cells are unstained. (G) Ear vesicle, junctional concentrations of a- catenin are indicated by arrows. (H) Ventral epidermis, cell membranes of the bilayered epidermis adjacent to the basal lamina exhibit only weak staining (arrows). en, endoderm; ep, epidermis; ev, ear vesicle; ey, eye vesicle; le, lens placode; nc, neural crest cells; no, notochord; nt, neural tube; pi, pigment layer; re, retinal layer; so, somites. Same histological procedure as in Fig. 7. Bars: 50 um.