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Fig. 5. Meis3 induces proliferative Iro3+ neural progenitors and inhibits their terminal differentiation. (A)Embryos were injected at the one-cell stage with Meis3-GR (250 pg; e-j), or VP16-Meis3 (250 pg; k-m) mRNAs, or Tsh1-MO (4 pmol; n-p) or Meis3-MOHypo (5 ng; q-s). Meis3-GR embryos were activated by 1M Dex at neurula stage 14, or kept untreated as controls. Iro3 and n-Tub expression was examined at neurula stages. The inset (n) shows Iro3 expression in early-stage wild-type embryos. u and w are magnifications of the hindbrain region (pink rectangle) of b and r, respectively. v and x show the trigeminal neuron of wild-type and Meis3- MOHypo embryos. Brackets and arrowheads indicate trigeminal expansion and ectopic differentiated foci, respectively. Cell proliferation was also assayed. Stacked images of 25 focal planes of cleared PH3-immunostained whole neural tubes are shown. Images are enlargements of the hindbrain region (pink rectangle in d). (t)Meis3- MOHypo phenotype frequency is shown. (y)Embryos were injected with Meis3-Myc (50 pg) and/or Meis3-MOHypo and western analysis at mid-neurula stages shows a moderate decrease in Meis3-Myc protein levels. (B)Transcriptional profiling of VP16-Meis3 neurons. RT-PCR to neural-specific developmental markers in mid-neurula embryos injected at the one-cell stage with VP16-Meis3 (250 pg) mRNA. (C)Meis3 represses p27Xic1 and Gadd45 expression cell- autonomously. RT-PCR to p27Xic1 and Gadd45 in mid- neurula stage intact or dispersed AC explants from embryos injected with VP16-Meis3 mRNA (0.8 ng) |
