Reintsch WE et al. (2005), beta-Catenin controls cell sorting at the notoc...

XB-ART-1499

J Cell Biol 2005 Aug 15;1704:675-86. doi: 10.1083/jcb.200503009.

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beta-Catenin controls cell sorting at the notochord-somite boundary independently of cadherin-mediated adhesion.

Reintsch WE , Habring-Mueller A , Wang RW , Schohl A , Fagotto F .

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In Xenopus laevis, patterning of the trunk mesoderm into the dorsal notochord and lateral somites depends on differential regulation of Wnt-beta-catenin signaling. To study the cellular requirements for the physical separation of these tissues, we manipulated beta-catenin activity in individual cells that were scattered within the trunk mesoderm. We found that high activity led to efficient cell sorting from the notochord to the somites, whereas reduced activity led to sorting in the opposite direction. Analysis of individual cells overexpressing beta-catenin revealed that these cells were unable to establish stable contacts with notochord cells but could freely cross the boundary to integrate within the somitic tissue. Interference with cadherin-mediated adhesion disrupted tissue architecture, but it did not affect sorting and boundary formation. Based on these results, we propose that the boundary itself is the result of cell-autonomous changes in contact behavior that do not rely on differences in absolute levels of adhesion.

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Species referenced: Xenopus laevis
Genes referenced: arvcf cdh3 ctnnb1 foxa4 gsk3b lrp5 myc myod1

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	Figure 1. Cells overexpressing β-catenin are excluded from the notochord. (A) Experimental set up and sketch of cross section shown in B. not, notochord. Arrowheads represent injections. (B–B′′′) Section of a β-catenin–myc-injected embryo stained for myc (B, red), cadherin (plasma membranes), and FoxA4 (notochord nuclear marker; B′, both in green) and counterstained with DAPI (B′′, blue). (B′′′) Overlay. Positive cells were found in the somites but not in the notochord. Arrows, notochord–somite boundary. Bar, 50 μm. (C) Merged image of a section stained for myc (red), cadherin, and the somitic nuclear marker MyoD (both green) and counterstained with DAPI (blue). (D) Merged image of a section from a control myc-eGFP–injected embryo stained as in B. Myc-eGFP–positive cells (red) distributed roughly equally between notochord and somites. (E) Quantitation of the distribution of β-catenin–myc- and myc-eGFP–expressing cells between notochord and somites. Numbers below represent total number of positive cells/number of embryos. Error bars represent SD. (F) Time course of β-catenin–myc expression analyzed by immunoblot (equivalent of one embryo/lane) compared with endogenous Gsk-3 (α and β isoforms).
	Figure 2. Cells overexpressing β-catenin sort during late gastrulation. (A) Dorsal explants of β-catenin–myc-injected embryos were fixed while flattened under a coverslip. (A′) Section of a stage 12.5 explant (anterior to the top) stained as in Fig. 1 B. Several β-catenin–myc-expressing cells can still be found in the notochord (arrows). Double arrows indicate wild-type cells in both tissues that tend to be elongated mediolaterally. (B) The percentage of positive cells in the notochord gradually decreases during late gastrula stages. Numbers below represent total number of positive cells/number of embryos. Error bars represent SD. Sketch depicts approximate changes in notochord shape. Arrow represents the anterior (a) to posterior (p) orientation of the notochord sketch. (C and D) Reduced FoxA4 expression (C and C′) and ectopic expression of MyoD (D and D′) in β-catenin–myc-expressing cells in the notochord (arrows). Sections (stage 12+) were stained as in Fig. 1 (B and C). (A′, C′, and D′) Arrowheads, notochord–somite boundary. no, notochord; so, somite. (E and E′) β-catenin–myc-expressing cell in the notochord shows reduced mediolateral elongation. (E) Stage 12+ section stained for myc (red), FoxA4, cadherin (green), and DAPI (blue). (E′) Sketch of E outlining cell shapes. Arrows, length and width of positive cell (red). Bars, 50 μm.
	Figure 3. Cells overexpressing β-catenin move from the notochord to the somites. Selected frames from time-lapse videos of dorsal explants with β-catenin–eYFP-expressing cells that were cultured from late gastrula on. (A) Cells 2 and 3 cross the boundary (dashed lines). (B) Higher magnification of a β-catenin–eYFP-expressing cell moving into the somitic mesoderm (arrowheads). Positive cell at the somitic side of the boundary (circles) shows no net movement. no, notochord; so, somitic mesoderm. Bars, 50 μm. See Videos 1 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200503009/DC1.
	Figure 4. The activity of Wnt signaling determines the direction of sorting. The pathway was activated using a constitutively active truncated LRP5 (A) or was inhibited using an axin variant (B). Sections (stage 13) were stained as in Fig. 1 B. Cells expressing LRP5-Flag sorted largely to the somitic mesoderm, whereas cells expressing myc-axin sorted to the notochord. Bar, 50 μm. (C) Quantitation of positive cell distributions. Numbers below represent total number of positive cells/number of embryos. Error bars represent SD.
	Figure 5. Signaling downstream of β-catenin is sufficient for sorting. Sections from stage 13 embryos were stained as in Fig. 1 B. (A) Cells expressing LEFVP16-myc sort to the somites. (B) Cells expressing LEFeng-myc do not sort but activate FoxA4 ectopically in the somites (arrows). (B′) Same section as B, only with FoxA4/cadherin staining. (C) Cells expressing myc–β-catenin–eng sort to the notochord. Bar, 50 μm. (D) Quantitation of positive cell distributions. Numbers below represent total number of positive cells/number of embryos. Error bars represent SD.
	Figure 6. Sorting to the notochord can be induced by expression of cadherin constructs. Sections of stage 13 embryos expressing the following myc-tagged constructs were stained as in Fig. 1 B: full-length C-cadherin (CadFL; A); CadΔcat, β-catenin–binding domain deleted (B); CadΔE, extracellular domain deleted (30 pg/injection; C); CadΔEΔcat, extracellular and β-catenin–binding domains deleted (D); and CadΔE (30 pg) and LEFVP16 (E). (A–D) Cells expressing cadherin constructs with β-catenin binding (A and C) sorted to the notochord. Bar, 50 μm. (E) Cells expressing both CadΔE (plasma membrane) and LEFVP16 (nuclear) sorted to the somites. (F) Quantitation of positive cell distributions, including experiments with higher amounts (50 pg) of CadΔE; see Fig. 7. Numbers below represent total number of positive cells/number of embryos. Error bars represent SD. (G) β-catenin binding of cadherin constructs; myc-eGFP (negative control) or cadherin constructs (all myc tagged) were immunoprecipitated from stage 11 extracts with anti-myc antibody. Precipitates were analyzed for β-catenin and Xarvcf by Western blotting. Input, equivalent of one embryo/lane. Immunoprecipitate (IP), equivalent of 20 embryos/lane.
	Figure 7. High CadΔE affects cell and tissue morphology. Embryos were injected with higher amounts of CadΔE-myc (50 pg/injection) alone (A–C) or together with LEFVP16-myc (D–F). All were stage 13 except for C (stage 12+). Staining as in Fig. 1 B. (A′ and D′) Stained only for FoxA/cadherin (green). (A and B) CadΔE-expressing cells are rounded, and the notochord morphology is perturbed (compare A with Fig. 1 D), but most positive cells are in the notochord and respect the boundary. Arrows in B point to two cells breaking the boundary. (C) Stage 12+ dorsal explant. On the left side, a normal boundary (inset a, dashed arrow) is surrounded by wild-type cells aligned mediolaterally (double arrows). On the right side, the boundary (inset b, dashed arrow) is disrupted by CadΔE-expressing cells, and the cell alignment is lost (asterisks). (D) Cells expressing CadΔE + LEFVP16 locate to the somites and respect the boundary. (E) Split notochord (arrowheads) surrounded by CadΔE/LEFVP16-positive cells. (F) Higher magnification of boundary (arrow). Positive cells are rounded but sort to the somites and respect the boundary. (G) Control, same magnification as F. myc-eGFP cells locate on both sides of the boundary (arrow) and have an elongated morphology. no, notochord; so, somite. Bars, 50 μm.

References [+] :

Brieher, Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps. 1994, Pubmed, Xenbase