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Figure. S3. Distribution of endogenous Notch 1 and β-catenin proteins at s7 in Xenopus embryos. Left and right halves of embryos were cut at the indicated Nieuwkoop and Faber stages (s) (left column, turquoise dotted boxes) and processed for whole-mount IF (A-D’’) or for IF in cryosections (E-N). The embryonic early axes were predicted according to the original pigmentation. (A) Bright field view. (B-B’’,E,J) Notch1 IF. (C-C’’,F,K) Total β-catenin IF. (D-D’’,G,L), merged images of Notch1 and total βcatenin IF. (B’-D’) Higher magnifications of the ventral region depicted by the white boxes in B-D. (B’’-D’’) Higher magnifications of the dorsal region depicted by the yellow boxes in B-D. Cells in the ventral-most side, with the highest Notch1 IF (white arrows, B',D’) stained weaker for nuclear β-catenin (white arrows, C') in comparison with cells of the dorsal side (yellow arrows, C’’,D’’), which have the lowest Notch1 IF (yellow arrows, B’’). (E, J) According to the DV orientation assigned by pigment distribution, Notch1 was significantly enriched in the ventral region in cryosections (8/9 embryos, P<0.0001; Mann-Whitney test; table S1). The pattern of total β-catenin is complex (F,G,I,K,L,N), with membrane-associated and cytoplasmic pools and some immunopositive nuclei (yellow arrows) that begin to be detected at this stage. We found embryos with a significant dorsal enrichment of total β-catenin (F) (4/9 embryos) and others with more ventral staining (K), according to pigment distribution (P<0.0001; Mann-Whitney test; see Table S1), although nuclear β-catenin begins to be detected in some dorsal nuclei (yellow arrows). One of the 9 embryos at s7 (Table S1) showed dorsal Notch1 enrichment and ventral total β-catenin enrichment, and this might be due to the inaccurate DV orientation assigned by pigment distribution; thus, this would render another embryo with ventral Notch1 and dorsal β-catenin. Notch1 is detected in ventral nuclei, which do not show strong nuclear β-catenin staining (white arrows), although some nuclei stain for both proteins (turquoise arrows). Note the lower total β-catenin IF in the apical region of the ventral-most cells, which shows instead high Notch1 IF (red arrows), consistent with the pattern in the whole-mount views, whereas in the embryo with dorsal β-catenin enrichment in E-I, there is higher β-catenin IF in the apical region of the dorsal cells (green arrows). d, dorsal; v, ventral; l, left; r, right. |
