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Fig. 3. Tsh1 is a Meis3 gene repressor that modulates Wnt/Beta-catenin activity and is regulated by Meis3 protein. (A,B)Meis3 expression in (Aa,b) mid-neurula and (Ba-d) gastrula embryos injected at the one-cell stage with Tsh1 mRNA (0.5 ng). (C)Meis3 expression in mid-neurula embryos injected at the one-cell stage with THVGR mRNA (40 pg; b) or Tsh1-MO (5 pmol; c), or both (d). Insets in c, d show Meis3 expression in late gastrula wild-type or THVGR- injected embryos, respectively. b' and d' schematically show Tsh1 modulation of the Wnt/Beta-catenin transcriptional response of Meis3. (D)Tsh1 expression in mid or early-neurula embryos injected at the one-cell stage with Meis3- MO (30 ng; b,e) or Meis3 mRNA (1 ng; c), or the Wnt3a-MO (45 ng; f). (E)RT-PCR to Tsh1 in mid- neurula stage AC explants from embryos injected with THVGR mRNA (40 pg) or Meis3-MO (30ng), or both. (F)RT-PCR to Tsh1 in mid-neurula embryos injected with Meis3 mRNA (0.8 ng) or Dkk1 mRNA (35 pg), or both. (G)Transcriptional kinetics of Tsh1 and Meis3 genes in response to increasing levels of Meis3 protein. RT-PCR was performed on pools of 18 mid-neurula AC explants from embryos injected with Meis3 mRNA (0.1-1.2ng). Results were quantitated and normalized to the Ef1-alpha control, and plotted as fold change. Bars are s.e.m. from three independent experiments. T samples showed no DNA contamination. A x-value is a non- injected control, with a y-value as a basal expression level. |
