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Fig. 1.
Distribution of endogenous Notch1 and β-catenin proteins from s1 to mid-blastula in cryostat sections of Xenopus embryos. Left halves of embryos were collected at the indicated stages and were then cut into 20 µm cryostat sections. The embryonic early axes were predicted according to the original pigmentation. (A-D) Notch1 IF was significantly higher in the ventral than in the dorsal region. This was confirmed by comparing vROI with dROI quantifications (P<0.0001, not shown). (A′-D′) Quantification of Notch1 IF of the embryos shown in A-D, respectively. Mean pixel intensity (MPI, y-axis) in a rectangular animal ROI was plotted (red line) in relation to the position in the DV axis (x-axis). The black line shows a linear estimate by linear regression analysis (parameters shown in inset): a positive slope indicates the increase of Notch1 IF from dorsal to ventral. The yellow arrows in B and C show Notch1 IF in the cell membranes between the animal dorsal and animal ventral blastomeres at s3 and s4, which correlate with the high peaks indicated with the blue arrows in B′ and C′, respectively. Note the ventral nuclei with highest Notch1 signal (D,P, yellow arrows), which correlate with the highest peaks in the profile in this s7 embryo (D′, blue arrows). (E-H) Total β-catenin IF from s1+ to s7. Although the IF in the images was low and noisy at the earliest stages (most evident in F,G), quantification in dROIs and vROIs in E-H (not shown) revealed that it was significantly higher in the ventral region in E (P<0.0001) and in G (P=0.0006), while there was no significant difference between the dorsal and the ventral regions in F (P=0.11). In the embryo shown at s7 (H), total β-catenin IF was significantly higher in the dorsal ROI (P<0.0001). (I-L) Merged images of Notch1 and β-catenin IF. Note the lower total β-catenin IF in the apical region of the ventral cells, which instead show high Notch1 IF (H,L, red arrows). Total β-catenin IF appears higher in the basal region of these cells and more uniform in the dorsal cells (H,L, green arrows) (see Fig. S4 for another example). (M-Q) Notch1 IF (M) and β-catenin IF (N) in an s8 embryo. (M′,N′) Highly contrasted images of M and N, respectively, merged with the DAPI nuclear image (Q) to facilitate visualization of Notch1+ nuclei on the ventral side (M′, yellow arrows) and β-catenin+ nuclei in the dorsal side (N′, yellow arrows). (O) Merged image of M and N, showing highest levels of Notch1 IF in the ventral side and highest levels of total β-catenin IF in the dorsal side in this s8 embryo. (P) Merged DAPI and Notch1 IF of s7 embryo. (R) Notch1 IF in the nuclear region was significantly higher in the ventral side. (S) β-Catenin IF in the nuclear region was significantly higher in the dorsal side. (T,U) Quantification of total Notch1 (T) and total β-catenin (U) in dorsal and ventral ROIs of the images shown in M and N, respectively. The ventral region expresses significantly higher levels of Notch1 protein than the dorsal region; in contrast, the dorsal region expresses significantly higher levels of β-catenin protein than the ventral region. Data are mean+s.e.m. ***P<0.0001 (double-tailed Mann–Whitney test). An, animal; d, dorsal; s, stage; v, ventral; Veg, vegetal. |
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Fig. 2.
The levels of Notch1 protein and mRNA and of CSL-dependent transcriptional activity are higher in the ventral side of Xenopus embryos. (A) Western blot of Notch1 in protein extracts from whole embryos and from dorsal and ventral halves at s3 and s7. At these stages, the more representative p100 band was quantified relative to GAPDH levels. Significantly higher levels of Notch1 protein were detected in ventral halves than in dorsal halves at both stages. *P<0.05 (t-test, from three independent experiments). Horizontal lines represent the mean values between the experiments; circles show the results for each experiment. (B-F′) Left and right halves of pigmented embryos were cut (B, dashed red line) at the indicated stages and processed as indicated for whole-mount IF of Notch1 (right halves; C-F), or for ISH of dorsal markers in contralateral left halves (wnt11 in C′-E′, chordin in F′). Notch1 IF is highest on the opposite side to the dorsal markers. The dotted white line in C,C′ shows the animal-vegetal axis. The dotted yellow circle in F′ shows the population of dorsal cells that are beginning to express chordin at this stage. (G-G′) The same albino two-cell embryo was processed for Notch1 IF and wnt11 ISH. Notch1 IF is shown in animal view before (G) and after (G′) ISH, and it is higher on the opposite side in relation to the early dorsal marker wnt11 (G′, arrows). (H-M) Left halves of embryos were cut at the indicated stages and processed for ISH of notch1. A higher expression of notch1 transcripts was observed in the ventral (white asterisks) than in the dorsal region (black asterisks). The arrows indicate the approximate dorsal and ventral limits of the notch1 domain. (N) RT-qPCR quantification of notch1 and wnt11 mRNA in dorsal and ventral halves of s5 embryos. Notch1 mRNA levels were significantly higher in the ventral halves, and wnt11 mRNA levels were significantly higher in the dorsal halves (*P<0.05, one-way ANOVA). (O) Notch reporter gene analysis of CSL-dependent activity. Relative luciferase/Renilla activity was significantly increased by nicd1 mRNA (NICD) and was significantly higher in the ventral half than in the dorsal half of s10 embryos. Data are mean+s.e.m. ***P<0.05 [one-way ANOVA analysis and Bonferroni's multiple comparisons test (Student's t-test)]. s, stage. |
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Figure. S1. Distribution of endogenous Notch 1 and β-catenin proteins from s1 through cleavage stages in Xenopus embryos (animal views). 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. The embryonic early axes were predicted according to the original pigmentation. (A,E,I,M) Bright field views. (B,F,J,N) Notch1 IF. (C,G,K,O) Total β-catenin IF. (D,H,L,P) Merged images of Notch1 and total β-catenin IF. We noted that in some early embryos, the highest levels of total β-catenin IF located relatively more dorsally (light blue asterisks) than the region were the highest levels of Notch1 IF were found (white asterisks) (see Table S2). d, dorsal; v, ventral; l, left; r, right. |
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Figure S2. Distribution of endogenous Notch 1 and β-catenin proteins from s1 through cleavage stages in Xenopus embryos (internal views). Left and right halves of embryos that were cut at the indicated Nieuwkoop and Faber stages (s) (left column, turquoise dotted boxes) and processed for whole-mount IF. The embryonic early axes were predicted according to the original pigmentation. (A,E,I,M) Bright field views. (B,F,J,N) Notch1 IF. (C,G,K,O) Total β-catenin IF. (D,H,L,P) Merged images of Notch1 and total β-catenin IF. We noted that in some whole-mount halves from early embryos, total β-catenin IF appeared to have a dorsal bias (light blue arrows) or its highest levels were apparently in a different place (light blue asterisk) in relation to the region were the strongest Notch1 IF was found (white asterisks) (See Table S2). d, dorsal; v, ventral; l, left; r, right; an, animal; veg, vegetal. |
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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. |
