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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. |
