Failure to proliferate and mitotic arrest of CDK11(p110/p58)-null mutant mice at the blastocyst stage of embryonic cell development

Abstract

The CDK11(p110) protein kinases are part of large-molecular-weight complexes that also contain RNA polymerase II, transcriptional elongation factors, and general pre-mRNA splicing factors. CDK11(p110) isoforms may therefore couple transcription and pre-mRNA splicing by their effect(s) on certain proteins required for these processes. The CDK11(p58) kinase isoform is generated from the CDK11(p110) mRNA through the use of an internal ribosome entry site in a mitosis-specific manner, suggesting that this kinase may regulate the cell cycle during mitosis. The in vivo role and necessity of CDK11(p110/p58) kinase function during mammalian development were examined by generating CDK11(p110/p58)-null mice through targeted disruption of the corresponding gene using homologous recombination. While heterozygous mice develop normally, disruption of both CDK11(p110/p58) alleles results in early embryonic lethality due to apoptosis of the blastocyst cells between 3.5 and 4 days postcoitus. Cells within these embryos exhibit both proliferative defect(s) and a mitotic arrest. These results are consistent with the proposed cellular functions of the CDK11(p110/p58) kinases and confirm that the CDK11(p110/p58) kinases are essential for cellular viability as well as normal early embryonic development.

FIG. 1.

Targeted disruption of the mouse cdc2l gene locus by homologous recombination. (A) Structure of the targeting vector and partial restriction map of cdc2l gene locus in mouse before and after homologous recombination. Exons are represented by vertical black boxes, and the position of the Southern blot probe (P) is indicated by the horizontal black box. Restriction enzymes: B, BamHI; X, XbaI; S, SacI; BX, BstXI. The mutated cdc2l allele was detected by a set of PCR primers: a, b, and c. The primers 1, 2, and 3 were used for the first round of the nested PCR amplification for blastocyst genotyping. (B) Southern blot analysis of CDK11^p110/p58 mutant ES cell lines. Genomic DNA isolated from ES cell clones following positive (G418) and negative (ganciclovir) selection were digested with BamHI and analyzed by Southern blotting using 5′ probe, yielding the predicted 5.8-kb band in addition to a 9.0-kb wild-type (WT) band. PCR amplification with primers d and e generated a 5.2-kb band containing the mutated allele. (C) Genotype analysis by Southern blot analysis (top) and PCR (bottom) of tail DNA of mice from heterozygote intercrosses. Tail DNA samples digested with BamHI were subjected to Southern blot hybridization with the 1.3-kb Neo probe, yielding a 1.7-kb band. The same DNA samples were also subjected to PCR with primers a and b for the WT allele and with primers a and c for the mutated (KO) allele, yielding amplification products of 180 and 320 bp, respectively. (D) The genotypes of the CDK11^p110/p58 embryos from CDK11^{p110/p58+/−} intercrosses were determined by nested PCR (see Materials and Methods). (E) Immunoblot analysis of CDK11^p110/p58 expression in WT and heterozygous targeted ES cell lines. Whole-cell lysates from a WT ES cell line (+/+) and targeted ES cell line (+/−) were analyzed by immunoblotting with the CDK11^p110-specific anti-P2N100 antibody. (F) Immunostaining of E3.5 CDK11^{p110/p58+/−} and CDK11^{p110/p58−/−} blastocysts with an affinity-purified C-terminal CDK11 polyclonal antibody, P1C, generated to a protein kinase domain shared by the CDK11^p110 and CDK11^p58 isoforms. The primary antibody was then detected with a fluorescein isothiocyanate-labeled secondary antibody (green, bottom panels) and indicates the presence of one or both of the protein kinases. The absence of a signal indicates that neither protein kinase is present in the blastocyst cells. To verify cellular integrity, we also stained blastocysts with DAPI, a reagent that specifically detects nuclear DNA (blue, top panels). White bar, 100 μm.

FIG. 2.

Morphological analysis of the outgrowths derived from the CDK11^p110/p58 blastocysts. Blastocysts were derived from CDK11^{p110/p58+/−} intercrosses, explanted at E3.5 (day 0), and grown in culture for 3 days, during which time they developed outgrowths. The CDK11^p110/p58+/+ (A and B) and CDK11^{p110/p58+/−} (C and D) blastocysts grew normally. In contrast, CDK11^{p110/p58−/−} (E and F) blastocysts stop further growth and development after E3.5. Magnification, ×11.2 for panels A through F. Embryos at E2.5 and their outgrowths after 4 days in culture in panels G through L. CDK11^p110/p58+/+ (G and H), CDK11^{p110/p58+/−} (I and J), and CDK11^{p110/p58−/−} (K and L) embryos are indistinguishable at E2.5. All images are not shown to scale (magnification, ×6.5 for day 4 wild-type and heterozygous outgrowth [panels H and J]; ×13 for panels G, I, K, and L). ICM, inner cell mass; TG, trophoblast giant cell.

FIG. 3.

CDK11^p110/p58 protein kinase deficiency resulted in apoptosis of the blastocyst cells. CDK11^p110/p58+/+, CDK11^{p110/p58+/−}, and CDK11^{p110/p58−/−} blastocysts were isolated at E3.5 from CDK11^{p110/p58+/−} intercrosses. The caspase 3 activity of the blastocysts was detected by the fluorescence of its covalently bound inhibitor, FAM-DEVD-FKM. Caspase 3 activity (B, D, and F) and nuclear DNA staining by DAPI (A, C, and E) were both examined by fluorescence microscopy. Bar, 50 μm.

FIG. 4.

Analysis of blastocyst cell apoptosis by TUNEL. CDK11^{p110/p58+/−} (A) and CDK11^{p110/p58−/−} (B) blastocysts were isolated at E3.5 from CDK11^{p110/p58+/−} intercrosses. After 12 h in culture, the fixed, permeabilized blastocysts were stained with TUNEL reagents (E and F) and DAPI (C and D). Bar, 50 μm.

FIG. 5.

Failure of the CDK11^{p110/p58−/−} blastocyst cells to proliferate normally. CDK11^p110/p58 blastocysts at E3.5 were recovered from CDK11^{p110/p58+/−} intercrosses and grown in culture in the presence of 10 μM BrdU for 16 h. The fixed blastocysts (panels A, E, and I) were then stained with anti-BrdU antibody (panels C, G, and K), an antibody specific to the M-phase-specific marker histone H3 phosphorylated at Ser¹⁰ (panels D, H, and L), and DAPI (panels B, F, and J). The genotype of each embryo was determined by using nested PCR. Representative CDK11^p110/p58+/+ (A to D), CDK11^{p110/p58+/−} (E to H), and CDK11^{p110/p58−/−} (I to L) blastocysts are shown. ICM, inner cell mass. Bar, 50 μm.

FIG. 6.

Representative confocal microscopy images of CDK11^p110/p58+/+ (A) and CDK11^{p110/p58−/−} mutant (B) embryos stained with DAPI (blue) and anti-PHH3 antibody (red). Bar,100 μm. Increased number of cells undergoing mitotic arrest observed in the CDK11^{p110/p58−/−} mutant embryos at E3.5 are shown. Mitotic arrest was determined by counting the number of cells positive for phospho-Ser-histone H3 (PHH3), the M-phase-specific marker. The percentage of PHH3-positive blastocyst cells from among the total number of blastocyst cells (determined by DAPI staining of cellular nuclei) is represented in the inset bar graph.