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Fig. 8. Xmxi1 induces proliferation in the open neural plate of Xenopus embryos. MT-Xmxi1 (500 pg), EnR-Xmxi1 (500 pg), and Xmxi1-GR (500 pg, induced at stage 10.5 with dexamethasone), Xmxi1-MO (12.5 ng), or the control-MO (Co-Mo) (12.5 ng) was injected in Xenopus embryos. Proliferation on the injected side compared with the noninjected side was measured by counting (A) pH3 or (B) BrdU-positive cells of 15 consecutive sections of five embryos in the open neural plate region. Shown is the average number of positive cells per section. Error bars indicate the standard error of the mean. (C) Forced cell cycle arrest does not alter the phenotype of Xmxi1-injected embryos. In Xmxi1-GR-injected embryos, proliferation was blocked by HUA treatment at stage 10 and induced at stage 10.5 with dexamethasone. (D) Inhibition of MT-XMxi1-induced apoptosis does not rescue loss of N-tubulin. Overexpression of MT-Xmxi1 (500 pg) led to an increase in apoptosis as seen by TUNEL staining (upper panel, right). Coinjection of human Bcl2 mRNA (500 pg) blocked apoptosis (lower panel, right) but did not influence the effects of MT-Xmxi1 on Sox3 and N-tubulin. |
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Fig. 2. (A–J) Regulation and requirement of Xmxi1 during primary neurogenesis. Whole-mount in situ hybridization of stage 14 embryos injected with Sox3 (50 pg), X-ngnr-1 (50 pg), NeuroD (500 pg), Notch-ICD (50 pg), or DN-Su(H) (300 pg), as indicated in the upper right hand corner. The antisense probes used are indicated in the lower left hand corner. The injected side (β-gal, light blue) is on the right, and embryos are shown as a dorsal view, anterior down. Red arrowhead marks ectopic expression (A, C) or the increased density of the lateral stripe (I, J). (K) Xmxi1 is activated by X-ngnr-1 and SoxD, but not by the neural inducer noggin. RT-PCR analysis of animal caps isolated from embryos injected with noggin (50 pg), X-ngnr-1 (25 pg), and SoxD (200 pg), as indicated. Histone H4 was used as a loading control. CC, control caps; CE, control embryos. (L, M) N-tubulin expression of stage 14 embryos injected with X-ngnr-1-MO (15 ng) and X-ngnr-1 (25 pg), as indicated in the upper right hand corner. (N) SoxD activation of Xmxi1 does not require X-ngnr-1. Animal caps were isolated from embryos injected with SoxD (200 pg), X-ngnr-1-MO (15 ng), X-ngnr-1 (25 pg), and control-MO (Co-MO) (15 ng), as indicated, cultured until sibiling embryos reached stage 14 and subject to real-time RT-PCR analysis. Expression levels were normalized to ornithine decarboxylase (ODC) and compared to the induction capacity of SoxD, which was set to 10. Note that X-ngnr-1 RT primers detect both endogenous and injected X-ngnr-1 RNA. |
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Fig. 5. Xmxi1 activates ectopic Sox3 and inhibits neuronal differentiation. (A–N) Whole-mount in situ hybridization of stage 14 embryos injected with 500 pg of MT-Xmxi1, MT-Xmad1, MT-Xmxi1-DBM, EnR-Xmxi1, or Max, as indicated in the upper right hand corner. Antisense probes used are indicated in the lower left hand corner. The injected side is always on the right, and all embryos are shown as dorsal views, anterior down with the exception of C and N, which are ventral views. |
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Fig. 7. (A) Xmxi1 induces neuronal differentiation in neuralized late, but not early animal caps. Animal caps were harvested from embryos injected with noggin (50 pg) and MT-Xmxi1 (500 pg), as indicated. At the equivalent of stage 14 or 20, the caps were harvested and analyzed by real-time RT-PCR. Expression levels were normalized to ODC and compared to control caps, which were set to 1. (B–E) The inhibition of neuronal differentiation by Xmxi1 is only transient. At tailbud stages, MT-Xmxi1 (750 pg) ectopic Sox3 is still present (B) and ectopic N-tubulin expression is also detected (D). (C, E) Transversal sections of B and D, respectively. Red arrowhead marks ectopic expression. |
