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ß-catenin, MAPK and Smad signaling during early Xenopus development

Anne Schohl and François Fagotto*

Department of Cell Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany


Figure 1
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  		Fig. 1. Example of image processing for the analysis of nuclear signals: ß-catenin staining of a parasagittal section, stage 10.25. 25 consecutive images of ß-catenin signal (A, red channel) and of DAPI staining (B, blue channel) were collected and collated automatically. The two images were merged (C), and filtered through a mask (D) created from the DAPI image. Because nuclei are small and their intensity difficult to visualize at low magnification, the nuclear area was expanded (E). To obtain semi-quantitative information, the intensity of the ß-catenin signal was translated into pseudocolors (F). The intensities were divided into five categories and translated into a color code (red to white) used for our drawings (G). Note that pale yellow can represent very weak or inhomogeneous signals. An identical procedure was used to analyze signals for P-Smad2 (red channel), P-Smad1 and P-MAPK (green channel).

 

Figure 2
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  		Fig. 2. Distribution of nuclear ß-catenin, P-MAPK and P-Smads during blastula stages. Because there are only few nuclei per section at early stages, nuclear images of several sections were superimposed (number of sections: stages 8 and 8.5=5; stage 9=3-4; stage 9.5=3). ß-catenin and P-MAPK images are from double-stained sections. Arrows and arrowheads point at particular aspects of the patterns discussed in the text.

 

Figure 3
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  		Fig. 3. Examples of staining at stage 9 (blastula). (A-C) Original images; (A'-C',A''-C'') nuclear images. ß-catenin and P-MAPK images are from double-stained sections. In the Smad1 image (A), the yolk was counterstained with Eriochrome Black (red). (C',C'') Nuclear images of 4 equatorial sections, superimposed. Arrows and arrowheads: prominent patterns, see explanations in the text.

 

Figure 4
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  		Fig. 4. ß-catenin distribution at mid (8.5) and late blastula (9.5) stages in normal and UV-irradiated embryos. (A-F) Original images; (A'-F') nuclear images. Arrows, arrowheads and asterisks: prominent patterns, see explanations in the text.

 

Figure 5
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  		Fig. 5. Examples of staining at early gastrula stage. (A-D) Original images; (A'-D',A''-D'') nuclear images. ß-catenin and P-MAPK images are from double-stained sections. bp, dorsal blastopore lip. Other symbols: see explanations in the text.

 

Figure 6
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  		Fig. 6. Examples of staining at various gastrula stages. (A-E) Original images; (A',C'-E',C''-E'') nuclear images. C'' and D'' are from a double-stained section. All sections are sagittal. bp, dorsal blastopore lip. Other symbols: see explanations in the text.

 

Figure 7
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  		Fig. 7. ß-catenin/P-MAPK staining of stage 16 neurula, parasagittal section. (A) Original ß-catenin image; (B) ß-catenin nuclear image; (C) P-MAPK nuclear image; (D) overlay of ß-catenin and P-MAPK nuclear images. Note the overlapping staining (asterisk) in the posterior circumblastoporal collar (cc) and the complementary patterns in the brain (arrowhead and carets). The arrow indicates the midbrain-hindbrain boundary. Star, posterior endodermal ß-catenin staining; en, endoderm; ep, epidermis; fb, forebrain; md, midbrain; hb, hindbrain; np, neural plate; sm, presomitic mesoderm.

 

Figure 8
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  		Fig. 8. Schematic drawings of the various stages and the main regions of the early Xenopus embryo.

 

Figure 9
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  		Fig. 9. Fig. 9, Fig. 10, Fig. 11, Fig. 12 are summary diagrams of ß-catenin, P-MAPK, Smad2 and Smad1 pattern during early development, respectively. Each drawing represents an average pattern obtained by analysis of several sections. The relative intensities between various stages were compared using pseudocolors images (see Fig. 1). Red is strongest, pale yellow is very weak/inhomogeneous. The spotted pattern of Smad2 at stage 8 indicates a very heterogeneous signal. All sections are sagittal unless stated otherwise. eq, equatorial; ps, parasagittal; tr, transversal.

 

Figure 10
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  		Fig. 10. MAPK signaling during early Xenopus development (see Fig. 9 legend).

 

Figure 11
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  		Fig. 11. Smad2 signaling during early Xenopus development (see Fig. 9 legend).

 

Figure 12
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  		Fig. 12. Smad1 signaling during early Xenopus development (see Fig. 9 legend).

 

Figure 13
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  		Fig. 13. Signaling patterns at blastula stage in manipulated embryos. (A-A''') controls; (B-B''') UV-irradiated embryos; (C-C''') LiCl-treated embryos; (D-D''') embryos ventrally injected with ß-catenin mRNAs (large arrowhead). (A-D,A''-D'') original images; (A'-D',A'''-D''') nuclear images. A-A'', B-B'' and C-C'' are from double stained sections. All other images are from contiguous sections from the same embryos. Arrows, arrowheads and asterisks: see explanations in the text.

 

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