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FIG. 5. Xebf2/Xcoe2, XNeuroD, and Xebf3 function in a unidirectional cascade during primary neurogenesis. Embryos were injected with RNA for XNeuroD (A, B), Xebf3 (C), and Xebf2 (F) at the two-cell stage and then assayed by whole-mount in situ hybridization at neural plate stages. Embryos are shown in a dorsal view with anterior to the bottom and injected side to the right (marked by X-gal staining). Overexpression of XNeuroD promoted ectopic expression of Xebf3 (A), while it suppressed the expression of Xebf2/Xcoe2 (B). Overexpression of Xebf3 did not affect Xngnr-1, XNeuroD, and Xebf2/Xcoe2 expression patterns (C), while Xebf2/Xcoe2-injected embryos showed ectopic expression of Xngnr-1, XNeuroD, and Xebf3 (F-H). (I) RT-PCR performed on animal caps isolated from XNeuroD (I), Xebf3 (L), or Xebf2/Xcoe2 (M) -injected embryos. The overexpression of XNeuroD caused a strong ectopic activation of Xebf3 (I, lane 2). Note that, in these caps, XNeuroD induced a very weak expression of Xebf2/Xcoe2 (I, lane 2). Overexpression of Xebf3 did not induce the expression of Xebf2 or XNeuroD (L, lane 2). In contrast, Xebf2-injected caps showed the activation of XNeuroD as well as Xebf3 (M, lane 2). Caps isolated from ﰁgal-injected embryos and RNA extracted from total embryos (TE) served as negative and positive controls (lanes 1 and 3). Muscle actin was used as a mesodermal marker, and EF1-alpha was used to normalize the samples. |
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FIG. 7. Xebf3 is insensitive to Delta/Notch-mediated lateral inhibition mechanism. Embryos were injected with RNA at the two-cell stage then collected at neural plate stages and assayed by in situ hybridization for expression of the neural markers N-tubulin and XNF-M, and of Xebf3 and Xebf2/Xcoe2, as indicated at the top. Injected embryos are oriented with their anterior side down (dorsal view, injected side revealed by X-gal staining). Overexpression of an activated form of the Notch receptor (Notch-ICD) caused a suppression of N-tubulin expression (C) and no change in XNF-M expression (D) on the injected side. XNF-M is normally turned on at stage 19. Embryos coinjected with Notch-ICD and Xebf3 (E, F) gave a similar pattern of ectopic N-tubulin and XNF-M expression as with Xebf3 alone (see Figs. 4C and 4D), suggesting that Xebf3 function was not inhibited by activated Notch. In contrast, embryos coinjected with Notch-ICD and Xebf2/Xcoe2 showed an inhibition of neurogenesis (G, H), as compared with embryos injected with Xebf2/Xcoe2 alone (Figs. 4E and 4F). The block of the Delta/Notch pathway induced by injecting X-Dl1stu did not influence Xebf3 expression (I) but caused an increase in the Xebf2/Xcoe2-positive neurons within the three stripes of primary neurons (L). When lateral inhibition was similarly blocked in XneuroD-injected embryos, overexpression of XNeuroD promoted ectopic expression of both Xebf3 and Xebf2/Xcoe2 (M, N). |
