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Abstract Radial glia cells function as guide cells for neuronal migration and a source of neural progenitor cells, and play a crucial role for the development of the central nervous system. To date, several signals have been demonstrated to promote the formation of radial glia cells and Notch signaling is one such signal. However, the mechanism of the signaling hierarchy of radial glia developmental cascade promoted by Notch signaling still remains incomplete. Here we show that Notch signaling promotes Xenopus radial glia formation and that the Notch activation is sufficient for radial glia formation prior to neural tube closure. Moreover, we have identified Oct-1 (POU2f1), a POU transcription factor, as a downstream target of Notch signaling by microarray based screen. We demonstrate that the expression of Oct-1 in the brain is regulated by Notch signaling and that Oct-1 is sufficient and necessary for radial glia formation. Together, Oct-1 is a downstream effector of Notch signaling during radial glia formation.
Fig. 1. X-Notch-1 overlaps the radial glia marker vimentin in Xenopus hindbrain. A–I: the expression of X-Delta-1 (A–C), X-Notch-1 (D–F) and vimentin (G–I) in embryos is shown. All panels are dorsal views, anterior toward left. A, D, G: stage 17, late neurula. B, E, H: stage 25, early tailbud. C, F, I: stage30, tailbud. A′–I′: transversal sections cut at the level of prospective hindbrain or hindbrain in panels A–I, respectively. a: anterior, p: posterior, e: eye, hb: hindbrain.
Fig. 4. Su(H) is sufficient but not required for radial glia formation. A–D: high magnification images of the injected and uninjected side of the hindbrain stained by anti-vimentin antibody in 250 pg at-GRSu(H)-injected (A), 500 pg dn-GRSu(H)-injected (B), GR-NICD-injected (C) and GR-NICD/dn-GRSu(H)-injected (D) embryos. Scale bar: 25 μm. E: RPratio between the injected and uninjected side of the hindbrain for each injection experiment. Gray bars or white bars indicate with or without DEX, respectively. *P < 0.001 compared with the ratio of each injection experiment without DEX. F: Whole-mount in situ hybridization analysis of XHey-1 expression in dn-GRSu(H)-injected embryos at stage 34 (n = 24/25). The injected side in each embryo is indicated by nucβ-Gal staining (red color).
Fig. 5. Notch signaling regulates the expression of Oct-1. A–C: the expression profile of Oct-1 in Xenopus embryo. All panels are dorsal views, anterior toward left. A: stage 17, late neurula. B: stage 25, early tailbud. C: stage 30, tailbud. A′–C′: transversal sections cut at the level of prospective hindbrain or hindbrain in panels A–C, respectively. a: anterior, p: posterior, e: eye, hb: hindbrain. D–M: whole-mount in situ hybridization analysis at stage 25. Oct-1 expression is expanded in the GR-NICD-injected (E: n = 33/45) and at-GRSu(H)-injected (L: n = 26/26) side of the brain indicated by a brace and reduced in the dn-Notch-injected (J: n = 17/30) but not dn-GRSu(H)-injected (M: n = 32/32) side of the brain. The activation of Notch at stage 20 did not expand the Oct-1 expression (F: n = 30/30). On the hand, the XHey-1 expression was increased or reduced by GR-NICD activated at stage 18 (G: n = 36/40) or dn-Notch (K: n = 30/42), respectively. The expression of vimentin was increased by GR-NICD (H: n = 32/39) and Oct1-GR (I: n = 34/40) activated at stage 18. The injected side in each embryo is indicated by nucβ-Gal staining (red color). The Oct-1 expression is not changed by injection of nucβ-Gal (D: n = 47/47).