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Fig. 4.
Notch1 is necessary for the expression of the ventral center gene wnt8a. (A-J) ISH expression pattern of wnt8a mRNA in embryos in which the Notch pathway was stimulated (B,D,F), or blocked (H,J), and in their corresponding sibling uninjected controls (A,C,E,I) or control Mo-injected sibling (G). Insets show the green fluorescence of the co-injected tracer DOG. (B,D) Embryos injected with 1 ng of nicd1 mRNA when the first cleavage was incipient. Note the upregulation and the dorsalwards expansion of wnt8a expression on the injected side in the dorsal view (B), whereas in the ventral view (D), where the tracer is more evenly distributed between the left and the right side of the embryo, wnt8a is strongly upregulated in comparison with the uninjected sibling control (C). (F) Embryo injected with 1 ng of notch1 FL mRNA at the one-cell stage. The expression of wnt8a was consistently stronger than in uninjected sibling controls (E). (G,H) Sibling embryos unilaterally injected with 40 ng of control Mo (G) or with 40 ng of Notch1 Mo (H) when the first cleavage was incipient. Note the downregulation of wnt8a towards the injected side in H. Wnt8a expression in control Mo-injected siblings was essentially unaffected. (J) Embryo injected with 2 ng of su(H)1DBM mRNA at the one-cell stage. The expression of wnt8a was consistently weaker than in uninjected sibling controls (I). (K) Summary of ISH results, expressed as the percentage of embryos showing the changes in wnt8a expression, indicated by the color codes [cyan, upregulated; orange, downregulated; gray, without changes (W/C)]. The number of total embryos analyzed (n) and the number of independent experiments (Exp) are indicated for each bar. (L) Quantification of wnt8a mRNA by RT-qPCR at s10 and at s11. Data are mean+s.e.m. *P<0.05 (one-way ANOVA analysis compared with control Mo injection). s, stage. |
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Figure S6. Rescue experiments showing the specificity of Notch1 Mo and that notch1 is necessary for the expression of other genes of the ventral program. (A) Uninjected control. (B-D) Embryos unilaterally injected at the 2-cells stage with 1 ng of nicd1 mRNA (B), 40 ng of Notch1 Mo (C), or with 40 ng of Notch1 Mo + 1 ng of nicd1 mRNA (D). (B’-D’) Fluorescence of the co-injected tracer DOG. The effect of the injections was assessed by comparing the expression pattern of wnt8a in the injected side (shown at the right side of the photographs) with the non-injected side (black arrowheads, B-D). nicd1 expanded wnt8a expression in the injected side in the dorsal region (compare green and black arrowheads). The down-regulation of wnt8a in the injected side by Notch1 Mo (red arrowhead, C) was rescued by co-injection of nicd1 mRNA (yellow arrowhead, D). (E) Summary of the rescue results analyzed by ISH, expressed as the percentage of embryos showing the changes in wnt8a expression indicated by the color codes. Cyan, up-regulated; orange, down-regulated; gray, without changes (w/c). The number of total embryos analyzed (n) and the number of independent experiments (Exp) are indicated below the bars. (F-J) RT-qPCR quantification at s11 of the following mRNAs: wnt8a (F), the bonafide Notch target hes9.1 (G), and the ventral markers trim29 (H), k81a1 (I), and tfap2a (J). Notch1 Mo significantly down-regulated all of these genes, whereas nicd1 mRNA completely rescued their down-regulation produced by Notch1 Mo. Results are represented as the mean+s.e.m. Statistical significant differences between means in relation to the Control Mo injection are indicated with asterisks (P<0.05; One-way ANOVA analysis). Nieuwkoop and Faber stages (s) are indicated in each panel. |
