To determine the effect of disrupting the balance of Cdk kinase and phosphatase activities in embryos prior to the MBT, 2.5 ng mRNA encoding wild-type Wee1 or luciferase (control) was microinjected into one-cell stage embryos. Western analysis confirmed expression of exogenous Wee1 throughout the developmental stages examined (Figure 1A). Embryos expressing exogenous Wee1 exhibited slower cleavage cycles. At 4.5 hours post-fertilization (hrs pf), embryos expressing exogenous Wee1 were delayed by approximately one cell cycle compared to controls embryos injected with luciferase mRNA (Figure 1B) (Stage 6 = 32 blastomeres compared to Stage 7 = 64 blastomeres) 26. Otherwise, these embryos developed with normal morphology through the MBT.

To determine whether the cell cycle lengthening induced by exogenous Wee1 coincided with the phosphorylation of Cdks, embryos expressing Wee1 or luciferase were assayed by Western analysis using a phosphoCdk primary antibody (Figure 1C). In untreated embryos, low-level tyrosine phosphorylation of Cdks occurs prior to the MBT then increases at the MBT concurrent with cell cycle lengthening 121927. Similarly, control embryos expressing luciferase demonstrated low levels of Cdk phosphorylation until the MBT. In contrast, Cdks were phosphorylated on tyrosine 15 as early as 3 hrs pf in embryos expressing exogenous Wee1, suggesting the cell cycle delay resulted from the inhibition of Cdks.

Mitotic cleavage cycles and nuclear cycles of DNA replication can be uncoupled in early X. laevis embryos 1. Therefore, we wanted to determine whether exogenous Wee1 also affected DNA replication in early embryos. Total DNA was isolated from embryos expressing Wee1 or luciferase, resolved by gel electrophoresis and stained with ethidium bromide. Embryos expressing exogenous Wee1 contained less DNA throughout early development, compared to luciferase controls (Figure 1D). However, the DNA content increased through the early development of embryos overexpresssing Wee1 indicative of continuing DNA replication. The rate of increase in DNA content was consistent with the lengthened cleavage cycles, suggesting that nuclear and cytoplasmic cycles were coordinated. To examine nuclear morphology, embryos were collected at several stages, sectioned and stained with DAPI. Representative fields from embryos collected at Stage 9 are shown in Figure 1E. Embryos overexpressing Wee1 contained interphase and mitotic nuclei with normal morphology. There was no loss of nuclei as seen in embryos overexpressing cyclin E 21, although there were fewer and larger cells in these embryos, consistent with the delayed cell cycles. These results indicate that the overexpression of Wee1 delays both nuclear and cleavage cycles, but these cycles appear to remain coupled.

In previous studies, we showed that X. laevis embryos expressing exogenous checkpoint kinases, Chk1 or Chk2, exhibited a similar cell cycle delay and premature Cdk phosphorylation 2829. Furthermore, embryos expressing exogenous Chk2 developed normally through neurulation and beyond, and exogenous Chk2 protects embryos form apoptosis induced by ionizing radiation 29.

Embryos overexpressing Wee1 developed normally through the MBT until early gastrulation (~Stage 10.5). However, during gastrulation, these embryos began to appear abnormal, exhibiting a loss of cellular attachment and overall organization (Figure 2A). The gross morphology of embryos expressing exogenous Wee1 appeared consistent with that of embryos that had undergone apoptosis in response to ionizing radiation 23, unreplicated DNA, or expression of dominant-negative Chk1/Chk2 429.

To determine whether the kinase activity of Wee1 was required for its disruption of development, embryos were microinjected with wild-type (WT) or kinase-dead (KD) Wee1 mRNA (plasmids provided by Paul Mueller, University of Texas, San Antonio) 16. The embryos expressing KD Wee1 developed normally through gastrulation (Figure 2B), indicating that kinase activity is required for the effects of exogenous Wee1.

To determine whether overexpression of Wee1 induced apoptosis, embryos were assayed for caspase activity in embryo lysates by the cleavage of exogenous human PARP protein 4. Embryos expressing exogenous Wee1 were positive for caspase activity at gastrulation (Stage 11; ~12 hrs pf) indicated by the presence of a cleaved PARP fragment (Figure 2C). These results were surprising since exogenous Chk1 and Chk2, lengthen cleavage cycles and promote phosphorylation to the same extent as Wee1, yet exogenous Chk1 and Chk2 kinases inhibit rather than promote apoptosis 429. Like the effects on gross morphology, the activation of caspases was dependent on Wee1 kinase activity as caspase activity was not detected in embryos expressing KD Wee (Figure 2D).

In X. laevis cell-free egg extracts, Wee1 accelerates apoptosis through interaction with an SH2 domain in the Crk-adaptor protein. Furthermore, Wee1 can restore apoptosis in extracts depleted of SH2 domain interactors 30. To determine whether Wee1 associates with Crk in developing X. laevis embryos undergoing apoptosis, Wee1 was immunoprecipated from embryo extacts and the immunoprecipitates were immunoblotted for Crk. Constant levels of Crk were detected even in lysates overexpessing Wee1 (data not shown). This data indicate an association between Crk and Wee1 in vivo, and that all available Crk may be complexed with endogenous Wee1.

In Xenopus embryos, the MBT delineates a point when a host of cell cycle remodeling events occur. Among these remodeling events are the degradation of maternal Cdc25A 12 and cyclin E 1931. In previous studies, we showed that overexpression of Chk1/Chk2 causes premature degradation of Cdc25A and delayed degradation of cyclin E 2229. Furthermore, the transient activation of Chk1 at the MBT is required for the degradation of Cdc25A 32. Since overexpression of Chk1, Chk2, and Wee1 all lengthen the cell cycle and trigger premature phosphorylation of Cdks, we wanted to investigate the effect of exogneous Wee1 on the timing of Cdc25A and cyclin E degradation, two hallmarks of the MBT.

Messenger RNA encoding wild-type Wee1 or luciferase (control) was microinjected into one-cell stage embryos, then embryos were collected at the indicated times and subjected to Western analysis of Cdc25A and cyclin E. Unlike Chk1/Chk2, overexpression of Wee1 resulted in delayed rather than accelerated degradation of Cdc25A (Figure 3A).

Additionally, overexpression of Wee1 delayed the degradation of cyclin E until mid-gastrulation (Figure 3B), similar to overexpression of Chk1 22 and the expression of exogenous Δ34-Xic1 9. Both Chk1 and Δ34-Xic1 inhibit cyclin E/Cdk2 in embryos and are the only reagents known to delay the timing of cyclin E degradation in X. laevis embryos. This evidence and the prediction of our mathematical model suggest that timing of cyclin E degradation is determined by cyclin E/Cdk2 activity itself 18. Therefore, we hypothesized that Wee1 may also inhibit cyclin E/Cdk2 in X. laevis embryos.

Inhibition of Cdk1 by Wee1 has been well characterized in X. laevis and other systems 10333435. In human HeLa cells, Wee1 also phosphorylates Cdk2, inhibiting entry into S-phase 232425. In order to determine whether Wee1 inhibits Cdk2 in early X. laevis embryos, cyclin E was immunoprecipitated from embryos overexpressing Wee1, and Western analysis was performed on the immunoprecipitates to detect inhibitory tyrosine phosphorylation on Cdk2. Embryos overexpressing Wee1 expressed higher levels of phosphorylated Cdk2 (PCdk) indicating inhibition of Cdk2 by Wee1 (Figure 3C). These results suggest that the delays in degradation of cyclin E and Cdc25A by the overexpression of Wee1 could be due to the inhibition of Cdk2. The inhibition of cyclin E/Cdk2 is also consistent with the reduced DNA content in embryos expressing Wee1, since cyclin E/Cdk2 promotes the initiation of DNA replication during S phase 3637.

To more directly measure Cdk activity, cyclin E immunoprecipitates were tested for kinase activity against histone H1. Immunoprecipatates were incubated with histone H1 and γ-³²P ATP, reactions were resolved by polyacrylamide gel electrophoresis, H1 bands were excised, and counted by scintillation counting (Figure 3D). Despite the increased tyrosine phosphorylation of Cdk2 in embryos overexpressing Wee1, Cdk2 kinase activity was not repressed. In fact, Cdk2 activity persisted in these embryos after it had decreased after the MBT in control embryos. The persistence of Cdk2 is consistent with the delay in cyclin E degradation (Figure 3B). However, it was surprising that Cdk2 activity was not reduced since we had observed tyrosine phosphorylation of Cdk2 (Figure 3C) and this phosphorylation event is known to inhibit Cdk2 activity 2836. One explanation may be that exogenous Wee1 only phosphorylates a fraction of Wee1. Whether this fraction represents a specific pool that is inhibited or represents a steady state of total Cdk2 is not known.

Overexpression of Wee1 in Xenopus embryos resulted in a post-MBT apoptotic death. Since the overexpression of Wee1 not only alters cell cycle remodeling events (Figure 3) and the concentration of DNA at the MBT (6 hr pf; Figure 1D), but also phosphorylates Cdk2 (Figure 3), we wanted to determine whether inhibition of Cdk2 contributed to the induction of apoptosis. In order to inhibit Cdk2 directly, one-cell stage embryos were injected with Δ34-Xic1 protein or p27Xic1CK- protein as a control. The Δ34-Xic1 protein is a truncated form of the full length-Xic1 that specifically inhibits the activity of cyclin E/Cdk2 in X. laevis 38. In X. leavis embryos, the expression of Δ34-Xic1 delays the degradation of cyclin E, thereby disrupting the cyclin E/Cdk2 timer 9. The p27Xic1CK- construct contains four point mutations in the Cdk2 binding site that inhibit p27Xic1/Cdk2 interaction, thus serving as a negative control.

Embryos expressing Δ34-Xic1 were delayed slightly (~1 cell cycle at the MBT) compared to p27Xic1CK- controls (Figure 4A) but developed otherwise normally through the MBT. However, prior to gastrulation, embryos expressing Δ34-Xic1 died by apoptosis, indicated by gross morphology (Figure 4A) and activation of caspases (Figure 4B). In contrast, embryos injected with p27Xic1CK- protein persisted through gastrulation and neurulated (Figure 4A). These data indicate that specific inhibition of Cdk2 results in apoptosis in Xenopus embryos.

Eggs from wild-type Xenopus laevis (Xenopus Express) were fertilized in vitro, dejellied in 2% cysteine in 0.1× MMR (0.5 mM HEPES, pH 7.8, 10 mM NaCl, 0.2 mM KCl, 0.1 mM MgSO₄, 0.2 mM CaCl₂, 0.01 mM EDTA), and maintained in 0.1× MMR. Embryos were staged 26 and subjected to manipulation. Embryos were injected at the one-cell stage with specific concentrations of Wee1 or luciferase mRNA dissolved in 25–30 nL TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). In other experiments, embryos were injected with Δ34-Xic1 38 and p27Xic1CK- protein diluted in buffer (20 mM Hepes, pH 7.5, 88 mM NaCl, 7.5 mM MgCl₂, 10 mM β-mercaptoethanol). Δ34-Xic1 lacks the first 34 amino acids of the p27Xic1 protein. p27Xic1CK- has the conserved residue in the kinase domain mutated (R33A, L35A, F65A, F67A). Embryos were observed with an Olympus SZX12 stereomicroscope and photographed with an Olympus DP10 digital camera.

Wee1 cDNA in pBluescript was kindly provided by Dr. Monica Murakami (National Cancer Institutes. Plasmids encoding Wee1 were linearized and used as templates to produce polyadenylated mRNA using the Ambion T3 mMessage mMachine in vitro transcription kit (Ambion). The control mRNA encoding exogenous luciferase protein was prepared as described previously 28. Experiments with kinase-dead and wild-type Wee1 mRNA were performed with constructs provided by Dr. Paul Mueller (University of Texas at San Antonio). In the kinase-dead mutant, the codon for lysine 239 was converted to the codon for arginine.

Embryos were lysed in EB buffer (20 mM HEPES, pH 7.5, 80 mM β-glycerophosphate, 15 mM MgCl₂, 20 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 mg/mL leupeptin, 1 mM microcystin). Samples were then resolved on SDS polyacrylamide gels), transferred to a nitrocellulose membrane and blocked in 3% nonfat dry milk in TBS- 0.1%Tween (tris-buffered saline- 20 mM Tris-Base, 0.14 M NaCl, pH-7.6) or 5% BSA in TBS – 0.1% Tween. Membranes were incubated in primary antibody against Cdc25A and cyclin E (gifts from Dr. James Maller), and Wee1 (1:1000) (Zymed), diluted in 5% BSA-TBS- 0.1% Tween overnight at 4°C. Membranes were washed then incubated in secondary antibody (Peroxidase-conjugated AffiniPure Donkey Anti-Rabbit IgG (Jackson Immuno Research Laboratories Inc.) 1:10,000 in TBS- 0.1% Tween. Immunoreactive proteins were detected by chemilluminescence using an ECL Plus kit (Amersham).

Embryos were injected with Wee1 or luciferase mRNA, collected at indicated times, and lysed in DNA digestion buffer (10 mM Tris, 100 mM EDTA, 50 μg/mL RNase, 0.5% SDS). Lysates were incubated at 37°C for 2 hrs. Proteinase K was then added to a final concentration of 100 μg/mL with continued incubation at 50°C for 4 hrs with slight intermittent manual agitation. Genomic DNA was phenol/chloroform extracted, and precipitated overnight at -20°C in 100% ethanol. Samples were resuspended in TE, and resolved on a 0.7% agarose gel, and ethidium bromide-stained DNA was visualized and photographed under UV illumination.

Embryos were collected at the times indicated, fixed in 4% paraformaldehyde, dehydrated through an ethanol series, cleared in CitriSolv (Fisher), embedded in paraffin, sectioned 7 μm thick, deparaffinized, rehydrated, and stained with 1 μg/ml DAPI. Serial sections were viewed and photographed on an Olympus AX70 fluorescence microscope equipped with a Color View 12 digital camera.

Embryos previously injected with Wee1 and luciferase mRNA or Δ34-Xic1 and p27Xic1CK- proteins were collected at desired stages (post-gastrulation), snap frozen on dry ice, and assayed for the cleavage of exogenous PARP 4. Specifically, embryos were homogenized in caspase extraction buffer (10 μl/embryo) (CEB: 80 mM β-glycerophosphate, 15 mM MgCl₂, 20 mM EGTA, 10 mM DTT). Lysates were incubated with 2 ng/mL recombinant human PARP (Alexis Biochemicals) at 27°C for 15 min, resolved on an SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. Anti-PARP (1:5000) and HRP-conjugated anti-rabbit (Cell Signaling Technology) (1:2000) diluted in 10% nonfat dry milk in PBS were used as primary and secondary antibodies, respectively. Both full-length and cleaved PARP proteins were detected by chemiluminescence using an ECL Plus kit (Amersham).

Embryos were injected with Wee1 or luciferase mRNA, collected at indicated times, and lysed in EB. Antiserum against cyclin E (gift from Dr. James Maller) was used to immunoprecipitate cyclin E/Cdk2 or antibodies against Wee1 (Zymed) were used to precipitate Wee1 and associated proteins. Embryos lysates were precleared with protein G Sepharose beads (Sigma, St. Louis, MO) for 30 min, then mixed with antibodies, and incubated overnight on ice. Protein G Sepharose beads were added the next day and incubated with the immunoprecipitates for 1 h with rotation. Beads were then washed twice in low-salt buffer (20 mM Tris, pH 7.4, 5 mM EDTA, 0.1% Triton ×-100, 100 mM NaCl) and twice in high-salt buffer (20 mM Tris, pH 7.4, 5 mM EDTA, 0.1% Triton ×-100, 1 M NaCl). Immunoprecipitates were then mixed with 2× gel loading buffer (1× = 0.6 mM Tris base, 2% glycerol, 3% SDS, 0.002% bromphenol blue) containing 10 mM n-ethylmaleimide, and resolved on an SDS-polyacrylamide gel. Western analysis of cyclin E precipitates was performed as described using a primary phospho-Cdk antibody (Cell Signaling Technology) diluted 1:1000 or a monoclonal Crk antibody (BD Biosciences) diluted 1: 5000 in 5% BSA-TBS-Tween and a secondary HRP anti-rabbit or anti-mouse antibody (Cell Signaling Technology) diluted 1:2000 in 5% BSA-TBS- 0.1% Tween.

For kinase assays, immunoprecipitates were washed twice in kinase buffer (20 mM HEPES, pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol), and then incubated 20 min at 27°C with 25 μl of kinase buffer containing 0.2 mg/ml bovine serum albumin, 0.5 mg/ml histone H1, 200 μM [γ-³²P]ATP [2 cpm/fmol]). Reactions were terminated with 25 μl of 2× gel loading buffer containing 25% β-mercaptoethanol, heated at 95°C for 2 min, and resolved by PAGE. The gels were stained with Coomassie Blue and dried. Incorporation of [γ-³²P]ATP was determined by Cerenkov scintillation counting of the excised histone H1 band.

Rosetta cells (Novagen) were transformed with pGEX vector encoding the GST-p27Xic1CK- containing 4 mutations in p27Xic1 (R33A, L35A, F65A, F67A). Bacterial pellets were resuspended in buffer × (50 mM Tris pH 8, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA) containing 1 tablet of Complete protease inhibitors (Roche) and 1 mM DTT. Cell suspension was incubated at room temperature with 100 μg/mL lysozyme for 20 min with stirring then for an additional five minutes with 1/20 volume 10% Triton ×-100. Cell suspension was then sonicated 2 × 30 seconds and centrifuged at 17,510 × g for 1 hr at 4°C. The supernatant fraction was loaded onto a column of glutathione-agarose beads (Sigma) previously equilibrated with 5 volumes of extraction buffer (50 mM Tris pH 8, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA). Supernatant was passed through the column twice. The column was then washed with 10 volumes of buffer A (50 mM Tris pH 8.0, 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM DTT) and 10 volumes of buffer B (20 mM Tris pH 8.0, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM DTT). p27Xic1CK- protein was eluted with 5 mL buffer × containing 100 mM glutathione (GSH;Sigma), pH 8.0. Purified p27Xic1CK- was then concentrated by buffer exchange using Amicon Ultra Ultracel low binding regenerated cellulose tubes (Millipore, Bedford, MA), resuspended in buffer (20 mM Hepes pH 7.5, 88 mM NaCl, 7.5 mM MgCl₂, 10 mM β-mercaptoethanol) and stored at -80°C.