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First published online 11 September 2008
doi: 10.1242/dev.024919

Development 135, 3389-3400 (2008)
Published by The Company of Biologists 2008
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Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2

Ande Satyanarayana1, Cyril Berthet1,*, Javier Lopez-Molina2, Vincenzo Coppola1, Lino Tessarollo1 and Philipp Kaldis1,3,{dagger}

1 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute-Frederick, Bldg. 560/22-56, 1050 Boyles Street, Frederick, MD 21702-1201, USA.
2 Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA.
3 Institute of Molecular and Cell Biology (IMCB), Proteos, 61 Biopolis Drive, 138673 Singapore.


Figure 1
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  		Fig. 1. The Cdk1Cdk2KI knockin construct. (A) Schematic showing the strategy used to generate the Cdk1Cdk2KI knockin construct (for details see Materials and methods). Exons (red arrowheads) are numbered. (B,C) Mouse embryonic stem (ES) cell clones positive for the Cdk1Cdk2KI locus were screened for lacZ expression (β-galactosidase staining, blue) (B) and by PCR (C). Exon1Cdk2-Cdk2 indicates endogenous Cdk2, whereas Exon1Cdk1-Cdk2 indicates that exon 1 was from Cdk1 and the remaining coding region from Cdk2. Three independent embryonic stem cell clones (1, 2, 3) are shown that had the Cdk1Cdk2KI locus correctly targeted. (D) Western blots and histone H1 kinase assays indicating the expression level of knockin Cdk2-HA (top panel) and Cdk2 (bottom panel), and the kinase activity of knockin HA-tagged Cdk2 (second panel from top) and of endogenous Cdk2 (third panel from top) in Cdk2-/- (lane 1), Cdk2+/+ (lane 2) and Cdk2+/+ Cdk1+/Cdk2KI (lane 3) MEFs.

 

Figure 2
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  		Fig. 2. Genetically relocated Cdk2 loses its meiotic function. (A) Testes from P90 Cdk2+/+ (Aa), Cdk2-/- (Ab), Cdk2+/+ Cdk1+/Cdk2KI (Ac) and Cdk2-/- Cdk1+/Cdk2KI (Ad) mice. (B) Ovaries from P90 Cdk2+/+ (Ba), Cdk2-/- (Bb), Cdk2+/+ Cdk1+/Cdk2KI (Bc) and Cdk2-/- Cdk1+/Cdk2KI (Bd) mice. (C-F) H&E-stained testes sections from P90 Cdk2+/+ (C), Cdk2-/- (D), Cdk2+/+ Cdk1+/Cdk2KI (E) and Cdk2-/- Cdk1+/Cdk2KI (F) mice. (G,H) Testes sections, stained for β-galactosidase, from Cdk2+/+ Cdk1+/Cdk2KI (G) and Cdk2-/- Cdk1+/Cdk2KI (H) mice. (I-L) H&E-stained ovary sections from P90 Cdk2+/+ (I), Cdk2-/- (J), Cdk2+/+ Cdk1+/Cdk2KI (K) and Cdk2-/- Cdk1+/Cdk2KI (L) mice. Representative pictures are presented. Scale bars: 20 µm in C-F; 50 µm in G-L.

 

Figure 3
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  		Fig. 3. Knockin Cdk2 fails to function in meiosis. (A,B) H&E-stained testes sections from P10 Cdk2+/+ (Aa), Cdk2-/- (Ba), Cdk2+/+ Cdk1+/Cdk2KI (Ab) and Cdk2-/- Cdk1+/Cdk2KI (Bb) mice. Testes sections, stained for β-galactosidase, from P10 Cdk2+/+ Cdk1+/Cdk2KI (Ac) and Cdk2-/- Cdk1+/Cdk2KI (Bc) mice. Apoptotic testes sections from P10 Cdk2+/+ Cdk1+/Cdk2KI (Ad) and Cdk2-/- Cdk1+/Cdk2KI (Bd) mice. (C,D) H&E-stained testes sections from P20 Cdk2+/+ (Ca), Cdk2-/- (Da), Cdk2+/+ Cdk1+/Cdk2KI (Cb) and Cdk2-/- Cdk1+/Cdk2KI (Db) mice. Testes sections, stained for β-galactosidase, from P20 Cdk2+/+ Cdk1+/Cdk2KI (Cc) and Cdk2-/- Cdk1+/Cdk2KI (Dc) mice. Apoptotic testes sections from P20 Cdk2+/+ Cdk1+/Cdk2KI (Cd) and Cdk2-/- Cdk1+/Cdk2KI (Dd) mice. Scale bars: 40 µm, except 100 µm in Ad,Bd,Cd,Dd.

 

Figure 4
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  		Fig. 4. Knockin Cdk2 expression is insufficient to rescue the Cdk2-/- meiotic defect. (A-D) Immunohistochemistry of Cdk1, Cdk2 and HA (Cdk2KI) on testes from 3-month-old mice. Sections stained for Cdk1: Cdk2+/+ (Aa), Cdk2+/+ Cdk1+/Cdk2KI (Ba), Cdk2-/- (Ca) and Cdk2-/- Cdk1+/Cdk2KI (Da). Sections stained for Cdk2: Cdk2+/+ (Ab), Cdk2+/+ Cdk1+/Cdk2KI (Bb), Cdk2-/- (Cb) and Cdk2-/- Cdk1+/Cdk2KI (Db). Sections stained for HA: Cdk2+/+ (Ac), Cdk2+/+ Cdk1+/Cdk2KI (Bc), Cdk2-/- (Cc) and Cdk2-/- Cdk1+/Cdk2KI (Dc). Arrows in Aa and Ba indicate leptotene and zygotene spermatocytes. Arrows in Ca and Da indicate cells with a pachytene-like morphology. Arrows in Ab indicate cells with zygotene morphology. Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. Genetically replaced Cdk2 retains its normal subcellular localization. (A,B) Immunofluorescence staining of Cdk2 and HA-Cdk2 in serum-starved and serum-stimulated Cdk2+/+ Cdk1+/Cdk2KI MEFs at different time points. Cdk2 and HA-Cdk2 localization was detected by rabbit anti-Cdk2 and rabbit anti-HA followed by Alexa Fluor 568-conjugated goat anti-rabbit antibodies (red; Ae-h, Be-h), with the nuclei counterstained with DAPI (blue; Aa-d, Ba-d). The images were captured with a laser confocal microscope (63x). These representative pictures are from one of the three independent stainings. Scale bar: 10 µm. (C) Line graph displaying the proliferation rate of Cdk2+/+, Cdk2-/-, Cdk2+/+ Cdk1+/Cdk2KI and Cdk2-/- Cdk1+/Cdk2KI MEFs. (D) Western blots showing the expression of Cdk2, Cdk4, Cdk1, cyclin A2, cyclin B1, cyclin D1, cyclin E1, HA-Cdk2, p27 (panels 1-9 from top, respectively), HA-Cdk2/cyclin E1 and HA-Cdk1/cyclin A2 co-immunoprecipitations (panels 11 and 12 from top, respectively), as well as the histone H1 kinase activity of Cdk2 and HA-Cdk2 (Cdk2KI) (bottom two panels) in Cdk2+/+ (lane 1) Cdk2-/- (lane 2), Cdk2+/+ Cdk1+/Cdk2KI (lane 3) and Cdk2-/- Cdk1+/Cdk2KI (lane 4) MEFs. Actin (panel 10) served as a loading control.

 

Figure 6
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  		Fig. 6. Loss of Cdk2 leads to premature transcriptional activation of Cdk1. (A,B) The β-galactosidase staining pattern in regenerating livers of Cdk2+/+ Cdk1+/Cdk2KI (Aa-d) and Cdk2-/- Cdk1+/Cdk2KI (Ba-d) mice at the indicated time points. (C,D) Immunohistochemical BrdU staining pattern in the regenerating livers of Cdk2+/+ (Ca,Da), Cdk2-/- (Cb,Db), Cdk2+/+ Cdk1+/Cdk2KI (Cc,Dc) and Cdk2-/- Cdk1+/Cdk2KI (Cd,Dd) mice at the indicated time points. (E) Bar chart showing the percentage of BrdU-positive liver cells at the indicated time points in regenerating livers of Cdk2+/+, Cdk2-/-, Cdk2+/+ Cdk1+/Cdk2KI and Cdk2-/- Cdk1+/Cdk2KI mice after 70% PH. The average and s.d. (error bars) were calcualted after counting a minimum of 3000 nuclei from several low-power fields per sample from each genotype. For each time point, four mice were analysed. Scale bar: 100 µm.

 

Figure 7
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  		Fig. 7. Transcriptional activation of Cdk1 during embryogenesis. (A-F) H&E (A',B',C',D',E',F') and β-galactosidase (A'',B'',C'',D'',E'',F'') staining patterns during different stages of embryogenesis in Cdk2+/+ Cdk1+/Cdk2KI mice. At each stage, four to five embryos were sectioned.

 

Figure 8
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  		Fig. 8. Transcriptional activation of Cdk1 in adult tissues. (A-H) The organs from four Cdk2+/+ Cdk1+/Cdk2KI mice were collected, sectioned and stained. H&E (A'-H') and β-galactosidase (A''-H'') staining patterns in brain (A), heart (B), skin (C), kidney (D), lung (E) thymus (F), spleen (G) and testes (H). (I) Western blot showing the expression level of knockin HA-Cdk2 (upper panel) and endogenous Cdk2 (bottom panel) in various adult organs collected from Cdk2+/+ Cdk1+/Cdk2KI mice. Scale bars: 100 µm, except 250 µm in H',H''.

 

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© The Company of Biologists Ltd 2008