Zhu H , Doherty JR , Kuliyev E , Mead PE .

NCI P30 CA21765 NCI NIH HHS , P30 CA021765-31S3 NCI NIH HHS , P30 CA021765 NCI NIH HHS

	Figure 3. Expression of CDK9, cyclin T2 and cyclin K during Xenopus development. A: Reverse transcription PCR analysis of the developmental expression of CDK9 and its cyclin partners T and K. RNA isolated from embryos at the indicated stages was used as a template for reverse transcription followed by PCR amplification of the target genes with specific oligonucleotide primers. CDK9, cyclin T2, and cyclin K are present as maternal transcripts and are expressed throughout early embryonic development. Mix.3 is expressed during late blastula and gastrula stages (boxed). B: Whole mount in situ hybridization for CDK9, cyclin T, and cyclin K during Xenopus development. Expression of CDK9, and its cyclin partners, is detected throughout the embryo at early gastrula stages. Stage-11 embryos were sliced in half (Stage 11 bisected) to reveal the staining pattern inside the embryo. Each of the genes is expressed broadly throughout the mesendoderm during gastrula stages. During gastrulation, the expression of CDK9 remains high while the levels of cyclin T2 and, more profoundly, cyclin K decline. At tailbud stage, high levels of expression of CDK9, cyclin T2, and cyclin K are detected in the head with low-level expression detected throughout the body.
	Figure 6. Endogenous Xenopus brachyury is down-regulated by ectopic expression of Mix.3. Embryos were injected in one blastomere at the two-cell stage with test mRNA together with a beta -galactosidase ( beta -gal) mRNA as a lineage tracer (stained red). In situ hybridization analyses for Xbra expression (stained blue/purple) were performed at early gastrula (Stage 10.5). Representative embryos for each group are shown; the number of embryos examined and the percentage that are the same as the representative are noted below. A: beta -gal alone (100%, n = 61). B,C: Mix.3. Ectopic expression of Mix.3 results in decreased Xbra expression on the injected side of the embryo (96% have decreased Xbra expression, n = 107/112). D: Mix.3 Delta 123. A mutant of Mix.3 that cannot interact with CDK9 maintains Xbra repression activity (100%, n = 21). E: CDK9. Expression of CDK9 alone does not disrupt Xbra expression in the marginal zone of the embryo (100%, n = 23). F: Mix.3 + CDK9. CDK9 expression does not block the repression of Xbra expression by Mix.3 (100%, n = 25). G: Cyclin K (100%, n = 22). H: Cyclin T2 (100%, n = 23). I: CDK9 + cyclin K (100%, n = 51). J,K: CDK9 + cyclin T2. Expression of CDK9 with cyclin T2 disrupts Xbra expression (89%, n = 40/45). L: Mix.3 + CDK9 + cyclin K (100%, n = 25). M: Mix.3 + CDK9 + cyclin T2 (100%, n = 44). N: Mix.3 Delta 123 + CDK9 + cyclin T2 (98%, n = 46/47).
	Figure 7. CDK9:cyclin complexes modulate the endoderm-inducing activity of Mix.3. RT-PCR analysis for endodermal markers was performed on RNA prepared from animal pole explants dissected from injected embryos. A: Analysis of early endoderm marker gene expression in animal pole explants. Ectopic expression of Mix.3 induces expression of the early endoderm genes Sox17 beta and Darmin. Co-injection of CDK9:cyclin T2 blocks Mix.3's endoderm inducing activity. Deletion of the CDK9-interaction domain from Mix.3 (Mix.3 Delta 123) eliminates the ability of cyclin T2 to repress Mix.3-mediated endoderm gene expression. The deletion mutant (Mix.3 Delta 123), when injected alone, induced endoderm markers. The inhibition of Mix.3-directed endoderm induction in animal pole explants requires the kinase activity of CDK9. Co-injection of a kinase-dead mutant of CDK9 (CDK9dead) with cyclin T2 did not block Mix.3 endoderm induction indicating that this is not an inhibitory effect of cyclin T2 alone. Cyclin K co-injected with CDK9 did not interfere with Mix.3 endoderm-inducing activity. B: RT-PCR analysis of late markers of endoderm differentiation. Animal pole explants were assayed at stage 36 for the late endodermal markers endodermin and intestinal fatty acid binding protein (IFABP). As demonstrated at earlier stages, CDK9:cyclin T2, but not CDK9:cyclin K, can block Mix.3-directed induction of endodermal markers. The inhibition of Mix.3 endoderm by CDK9:cyclin T2 is dependent on the kinase activity of CDK9. C: The effect of Mix.3, CDK9, and cyclin T2 or K co-injection on endogenous darmin expression was measured by in situ hybridization. Embryos were injected in one blastomere at the two-cell stage with test mRNA together with a beta -galactosidase ( beta -gal) mRNA as a lineage tracer (stained red). In situ hybridization analyses for darmin expression (stained blue/purple) were performed at stage 18. The injected side (stained red) is on the left; endogenous darmin expression is seen on the untreated (right) side of the embryo. Representative embryos for each group are shown; the number of embryos examined and the percentage that are the same as the representative are noted below. (i) beta -gal alone (n = 61). (ii) Mix.3 induces ectopic darmin expression (white arrow) in the whole embryo (100% of embryos have ectopic darmin expression, n = 51). (iii) Mix.3 Delta 123. Deletion of the CDK9 interaction domain has no effect on the ability of Mix.3 to induce ectopic darmin expression (93% displayed ectopic darmin expression, n = 25/27). (iv) Mix.3 + CDK9 induces ectopic darmin expression similar to Mix.3 mRNA alone (98%, n = 45/46). (v) Mix.3 + cyclin T2. Cyclin T2, in the absence of added CDK9, does not block the ability of Mix.3 to induce ectopic darmin expression (100%, n = 27). (vi) Mix.3 + cyclin K. Mix.3 induces ectopic darmin expression in the presence of added cyclin K (100%, n = 20). (vii-viii) Mix.3 + CDK9 + cyclin K. Mix.3 induces ectopic darmin expression in the presence of added CDK9 and cyclin K (96%, n = 47/49). (ix-x) Mix.3 + CDK9 + cyclinT2. Co-injection of cyclin T2 mRNA with Mix.3 + CDK9 blocks ectopic darmin expression (86% showed inhibition of ectopic darmin expression, n = 49/57). (xi-xii) Mix.3 Delta 123 + CDK9 + cyclin T2. The ability of CDK9 + cyclin T2 to block Mix.3-directed ectopic darmin expression is dependent on the CDK9 interaction domain in the C-terminus of Mix.3 (94%, n = 29/31).

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