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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. |
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Figure. S4. Distribution of endogenous Notch 1 and β-catenin proteins in Xenopus mid blastula (internal views). Internal views of right (A-D’’) and left (F-O´) halves of embryos that were cut at s8 (as shown in the left column, turquoise dotted boxes) and processed for whole-mount IF. The embryonic early axes were predicted according to the original pigmentation. (A,F,F’,K,K’) Bright field views. (B-B'',G,G’,L,L’) Notch1 IF. (C-C'',H,H’,M,M’) Total β-catenin IF. (D-D'',I,I’,N,N’) Merged images of Notch1 and total β-catenin IF. (E-E'',J,J’,O,O’) Nuclear Hoechst staining. The vertical yellow dotted lines in (B-E) indicate the photocomposition of images taken at two slightly different focal planes to keep nuclei in focus in the dorsal and ventral region of the embryo. (B'-E') Higher magnification of the ventral region of the images shown in B-E, corresponding to the area indicated by the left red box in E. (B''-E'') Higher magnification of the dorsal region of the images shown in B-E, corresponding to the area indicated by the right red box in E. (F-J) and (K-O) Ventral and dorsal regions, respectively, of the same embryo. (F’-J’) and (K’-O’) Higher magnifications of F-J and K-O, respectively, corresponding to the yellow boxed area in F and K. Notch1 protein is more enriched in the ventral (white asterisks) than in the dorsal region (yellow asterisks) whereas the opposite occurs with nuclear β-catenin (white arrows). These patterns were consistently found both in right and left halves at this stage in embryos from different batches (94%, n=18, 3 batches of embryos; see Table S2). Interestingly, amongst the ventral cells, we could distinguish Notch1+ nuclei in several cells (G’,I’,J’, light blue arrows) while others did not appear to have nuclear Notch1 accumulation (G’,I’,J’, pink arrows). Bl, blastocoel cavity; d, dorsal; v, ventral; l, left; r, right; an, animal; veg, vegetal. |
