Fagotto F and Gumbiner BM (1994), Beta-catenin localization during Xenopus embryo...

XB-ART-20487

Development 1994 Dec 01;12012:3667-79.

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Beta-catenin localization during Xenopus embryogenesis: accumulation at tissue and somite boundaries.

Fagotto F , Gumbiner BM .

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beta-catenin is a cytoplasmic protein associated with cadherin adhesion molecules and has been implicated in axis formation in Xenopus (McCrea, P. D., Brieher, W. M. and Gumbiner, B. M. (1993) J. Cell Biol. 127, 477-484). We have studied its distribution in Xenopus embryos by immunofluorescence on frozen sections. Consistent with its function in cell-cell adhesion, beta-catenin is present in every cell. However, high levels are expressed in certain regions and different tissues of the embryo. No simple correlation appears to exist between the levels of beta-catenin with the expected strength of adhesion. High levels of beta-catenin were found in regions undergoing active morphogenetic movements, such as the marginal zone of blastulae and gastrulae. This suggests that high expression of beta-catenin could be involved in dynamic adhesion events. Surprisingly, beta-catenin also accumulates on plasma membranes that probably do not establish direct or strong contacts with other cells. In particular, high amounts of beta-catenin are found transiently at boundaries between tissue anlagen and at the intersomitic boundaries. This unexpected pattern of beta-catenin expression raises the possibility that this molecule participates in developmental processes, perhaps independently of its classical role in cell-cell adhesion.

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Species referenced: Xenopus
Genes referenced: cdh3 ctnnb1 mtor

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	Fig. 1. b-catenin distribution in cleavage stages and early gastrula. (A) Morula (stage 32 cells); (B) late blastula (stage 9), and (C,D) early gastrula (stage 10). b-catenin is present along the blastocoel roof (large arrows), but not the blastocoel floor (small arrows). The arrowhead in panel C shows the apex of the bottle cells on the dorsal side. (D) Detail of the dorsal marginal zone and of the bottle cells of the dorsal blastoporal lip from figure C. Small arrowheads show the superficial cells of the marginal zone. The large arrowhead points at the apex of the bottle cells, and the arrows show their swollen bases. (E) Control section from the marginal zone of a late blastula embryo incubated with a non-immune rabbit serum. Bars = 200 mm (A-C, E) and 100 mm (D). an, animal pole; vg, vegetal pole; mz, marginal zone.
	Fig. 2. Distribution of b-catenin in membrane and soluble fractions and in dorsal vs. ventral and animal vs. vegetal hemispheres. (A-C) 32- cell-stage embryos were fractionated by differential centrifugation as described in Materials and Methods. Three fractions, a low speed pellet containing the yolk and some plasma membranes (y), a high speed pellet containing all the membranes (m), and the corresponding supernatant (s) were analyzed by Western blot for (A) b-catenin, (B) a-catenin and (C) C-cadherin. Most of these three proteins were recovered in the membrane fraction. The supernatant contained small amounts of both catenins, but no C-cadherin. (D,E) 48-cell-stage embryos were dissected into halves and analyzed for b-catenin by Western blot. Aliquots equivalent of 2 halves of embryo were loaded per lane. an, animal; d, dorsal; v, ventral; vg, vegetal.
	Fig. 3. b-catenin localization in gastrula and neurula stages. (A) Sagittal section, stage 12; the large arrow shows the strong labeling in the deep involuting cells of the dorsal marginal zone. (B) Sagittal section, stage 13; large arrow: dorsal involuting marginal zone; small arrows: mesoderm-archenteron roof boundary. (C) Detail of the dorsal involuting marginal zone at stage 121/2, before involution (arrowheads) and after involution (arrows). (D) Detail of the dorsal and ventral blastoporal lips at stage 13; the arrows show the apical staining of the superficial involuting cells. (E) Transverse section, stage 17; the arrows indicate the mesoderm-endoderm boundary. ar, archenteron roof; bc, bottle cells; dl, dorsal lip of the blastopore; ec, ectoderm; en, endoderm; lm, lateral mesoderm; no, notochord; nu, neural plate; vl, ventral lip of the blastopore; yp, yolk plug. Bars = 200 um (A,B,E) and 100 um (C,D).
	Fig. 4. Distribution of catenins and C-cadherin in axial structures. (A-C) b-catenin staining of transverse sections. (A) Early neurula (stage 14+); (B) midneurula (stage 17); (C) early tailbud (stage 21). Large arrows point at the b-catenin-enriched tissue boundaries. Small arrows in panel C show the absence of b-catenin around the notochord at stage 21. (D-F) Transverse sections of stage 17 embryos stained for C-cadherin (D), a-catenin (E), and with a control, non-immune serum (F). Large arrows indicate accumulation of C-cadherin and a-catenin at tissue boundaries. (G) b-catenin accumulation at the boundary between somitic mesoderm (so) and endoderm (en) in a stage 17 embryo. (H-I) Sagittal sections through the notochord (no) of a stage 28 embryo stained for b-catenin (H) and a-catenin (I). (J-K) Controls for b-catenin staining: transverse sections of stage 16 embryos were stained using anti-b-catenin serum preincubated for 1 hour with 50 mg/ml GST-b-catenin fusion protein (J) or GST protein alone (K). ec, ectoderm; en, endoderm; no, notochord; nu, neural plate and tube; so, somitic mesoderm. Bar, 100 um.
	Fig. 5. Parasagittal section of a stage 24 tailbud embryo stained for b-catenin. cg, cement gland; ec, ectoderm; en, endoderm; nu, neuroderm; pm, posterior mesoderm; so, somites; small arrows, boundary bewteen the presomitic mesoderm and the endoderm; long arrow, boundary between a newly formed somite and a forming somite undergoing rotation; short arrows, intersomitic boundary between older somites; arrowhead, increased staining in the inner layer of the ectoderm. Bar, 200 um.
	Fig. 6. Catenin distribution in longitudinal sections of somites. Longitudinal sections of somites from stage 28 (A,B) and 41 (C) embryos stained for b-catenin (A,C) and a-catenin (B). (D) Control section (stage 28) labeled with a non-immune serum. (E-F) Control sections (stage 25) stained with anti-b-catenin serum preincubated for 1 hour with 5 mg/ml GST-b-catenin fusion protein (E) or 50 mg/ml GST protein alone (F). Large arrows, intersomitic boundaries; small arrows, lateral membranes of myocytes; ec, ectoderm. Bars, 100 um.
	Fig. 7. b-catenin distribution in transverse sections of late tailbud and early tadpole stages. (A) Stage 28; (B) stage 35. ca, cardiac tissue; ec, ectoderm; en, endoderm; no, notochord; nu, neural tube; so, somites; arrowheads, inner layer of the ectoderm. Bars, 200 um.
	Fig. 8. b-catenin distribution in the ectoderm. (A) Stage 14+; (B) stage 28; inserts show controls where the anti-b-catenin serum was preincubated with 50 mg/ml GST-b-catenin fusion protein (higher insert) or 50 mg/ml GST protein alone (lower insert); (C) stage 41; (D) cement gland at stage 24. ie, inner ectodermal layer; oe, outer ectodermal layer; arrowheads, basal membrane of the inner ectodermal layer; short arrows, apical junctions; long arrow, interface between the inner and outer layer. Bar, 50 um.
	Fig. 9. b-catenin distribution in activin-treated animal caps. (A) b-catenin staining of the elongated portion of an activin-treated animal cap fixed at stage 18 (staged from control embryos). b-catenin accumulates along the notochord-paraxial mesoderm (arrowheads) and the neuroderm-mesoderm (arrows) boundaries. (B) Consecutive section stained with Tor 70, a marker of the notochord. (C) b-catenin staining of a larger region of an activin-treated animal cap (stage 18) showing an accumulation of b-catenin in the inner ectodermal layer (arrows). (D) Section of an untreated animal cap (stage 18) stained for b-catenin. b-catenin is distributed homogeneously over the whole tissue. ec, ectoderm; me, mesoderm; no, notochord; nu, neuroderm. Bar, 100 um.