Int J Dev Biol
January 1, 2008;
Concentrations of TATA box-binding protein (TBP)-type genes affect chordamesodermal gene expression.
The TATA box-binding protein (TBP
) is an essential component of transcription initiation complexes of all three eukaryotic RNA polymerases. Recent knockdown studies revealed that basic transcription factors are essential not only for gene transcription but also for regulating specific gene expression. However, the mechanism of and the effect by regulation of TBP
expression are unknown during early embryogenesis. Here we show that the alteration of concentration of each TBP
-type gene affected mutually one another''s expression, suggesting that an optimal ratio of concentrations of TBP
-type genes induce expression of specific genes.
Int J Dev Biol
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Fig. 1. Over-expression and repletion of TBP inhibits axis formation. (A) Both over-expression of TBP and high-dose injection of TBP-MO into the future marginal zone region of all blastomeres of the 4-cell embryo reduced expression of Xbra. (B) Control embryo (st. 30). (C) Over-expression of TBP (700 pg/blastomere) into the future marginal zone region of dorsal blastomeres of the 4-cell embryo interfered with axis formation, including a short axis and absence of most of the head. (D) Over-expression into the ventral blastomeres of the 4-cell embryo had no effect on embryogenesis. (E) High-dose injection of TBP-MO (10 ng/blastomere) into the marginal zone region of dorsal blastomeres of 4-cell embryo also interfered with axis formation. (F) High-dose injection of TBP-MO into the ventral blastomeres of the 4-cell embryo had no significant effect on embryogenesis. (G) Over- expression of TBP reduced expression of chordamesodermal genes (center lane) at gastrula stage (st. 10). High-dose injection of TBP-MO reduced expression of all marker genes except for Xnot, which was increased by injection of TBP-MO. (H) Results of a luciferase assay using the reporter gene containing the promoter of goosecoid are shown. The luciferase activity was decreased by both over-expression of TBP and injection of TBP-MO at stage 10.
Fig. 4. The confirmation of the speci- ficity of each morpholino. TBP-MO, TBP2-MO and TLF-MO reduced spe- cifically translation of GFP containing sequences targeted with TBP-MO, TBP2-MO and TLF-MO at the 5region, respectively. GFP-TBP (250pg), GFP- TBP2 (250pg) or GFP-TLF (250pg) with Alexa594-dextran (300pg) was injected in the animal pole of 4-cell embryo. The fluorescent signals were observed at stage 10. Pictures on top panels indi- cate the injected embryos. Pictures on center panels indicate tracer signals with Alexa594-dextran. Pictures on bottom panels indicate GFP signals. Each MO reduced translation of each corresponding GFP construct, but did not reduce translation of the GFP con- struct containing different targeting sequence.