Cdk8 is essential for preimplantation mouse development

Abstract

The Cdk8 kinase and associated proteins form a nonessential transcriptional repressor module of the Mediator in the budding yeast Saccharomyces cerevisiae. Genetic analyses of this module have demonstrated functions ranging from environmental responses in budding yeast to organogenesis and development in worms, flies, and zebrafish. Here we have investigated the function of mammalian Cdk8 using mice harboring a gene trap insertion at the Cdk8 locus inactivating this kinase. No phenotypes were noted in heterozygote Cdk8+/- mice, but intercrossing these did not produce homozygous Cdk8-/- offspring. Developmental analysis demonstrated a requirement for Cdk8 prior to implantation at embryonic days 2.5 to 3.0. Cdk8-/- preimplantation embryos had fragmented blastomeres and did not proceed to compaction. As Cdk8 deficiency in cultured metazoan cells did not affect cell viability, the results suggest that transcriptional repression of genes critical for early-cell-fate determination underlies the requirement of Cdk8 in embryogenesis.

FIG. 1.

Characterization of the Cdk8 gene trap allele. (A) Schematic representation of the gene trap insertion in the Cdk8 locus. En2, SA, and pA indicate the engrailed 2 sequence and its splice acceptor and the polyadenylation site of the gene trap vector. (B) Anti-β-galactosidase Western blot analysis showing the expression of the Cdk8-β-Geo mutant protein in the targeted (RRS314) ES cells. (C) PCR genotyping of Cdk8^+/+ and Cdk8^+/− ES cells. (D) Southern blotting using a 5′ flanking probe of KpnI-digested DNA samples of Cdk8^+/− and Cdk8^+/+ mice. (E) Map of the determined genomic structure of the Cdk8 gene trap allele. B (BglI) and K (KpnI) designate restriction sites used for Southern blotting analysis. The locations of the probes used in the Southern blots are indicated by the black bars. (F) Cdk8 expression is indicated by LacZ staining of paraformaldehyde-fixed embryos extracted at E11.5. The indicated genotypes were obtained by PCR genotyping of the yolk sac. wt, wild type.

FIG. 2.

Cdk8 is required at E2.5. (A) Embryos isolated at E2.5 of Cdk8^+/− intercrosses were genotyped using nested PCR. E2.5 is the last stage of embryogenesis where Cdk8^−/− embryos can readily be isolated. Molecular size markers are indicated on the right. (B) Phase-contrast photomicrographs of Cdk8^+/+ and Cdk8^−/− embryos at E2.5. Black and white arrowheads indicate fragmented blastomeres.

FIG. 3.

Proliferation of cultured metazoan cells is unaffected by Cdk8 depletion. (A) Efficiency of the Cdk8 knockdown (Cdk8 dsRNA) compared to that of the control (GFP dsRNA) in Schneider S2 cells was assessed by anti-Cdk8 Western blot analysis. (B) The proliferation rate of parallel S2 cultures was determined and is presented as population doublings (PD) over 11 days. (C) Anti-Cdk8 Western blot analysis of shRNA-mediated knockdown in transiently transfected 293FT cells. (D) The effect on proliferation of expressing shRNA-targeting Cdk8 in 293FT cells is determined as the number of drug-resistant colonies at 13 days posttransfection. The values are the average number ± SD of colonies/10 microscopic fields (10× objective, approximately 3.1 cm²) of three independent transfections normalized to pCMV-β-gal activity.