Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Abstract

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.

Figure 1. dwee1 Regulates Cdk1 during Syncytial Blastoderm Cycles

Cyclin B immunoprecipitates from extracts of syncytial blastoderm stage embryos were analyzed for associated Cdk1 and kinase activity toward histone H1 (H1). (A) Cyclin B immunoprecipitates were processed to detect phosphorylation of H1 (H1 ³²P) and the amount of H1 added to each reaction (Total H1). Similar amounts of Cdk1 coprecipitated with Cyclin B in wild-type and dwee1 extracts. The percentage of each H1 kinase reaction analyzed is indicated above each lane. M = mock immunoprecipitations from which anti-Cyclin B antibody was omitted. (B) Quantification of ³²P incorporation into H1 in (A). Data from three independent experiments are shown. In each case, ³²P signal from dwee1 mutant immunoprecipitate (dwee1) was normalized to the ³²P signal from wild-type immunoprecipitate (WT). Cyclin B-associated kinase activity toward H1 is 4.4-fold higher in precipitates from dwee1 extracts (p < .05). (C) Cdk1 is present at similar levels in extracts from wild-type and dwee1 mutant embryos. Western blotting for γ-tubulin confirms equal loading (10% input shown). (D) Cyclin B immunoprecipitates were incubated with GST-dwee1, GST, or buffer (control) before being processed for H1 kinase activity. Relative amounts of Cdk1, GST-dWee1, GST, and histone H1 in each reaction are shown. (E) Quantification of ³²P incorporation into H1 in (D). Data from each of three independent experiments were expressed as percent of buffer control. GST-dWee1 significantly inhibited Cdk1: Cyclin B kinase activity (p= .001). (F) Detection of pY15-Cdk1 by a phosphospecific antibody is phosphatase sensitive. Wild-type embryos in interphase of cycles 10–12 were treated with either phosphatase inhibitors (“inh +,” lane 1) or phosphatase (“ppase +,” lane 2). After being probed with anti-pY15-Cdk1 antibody, blots were stripped and reprobed with nonphosphospecific anti-cdk1 as a loading control. (G) dwee1 mutants have reduced levels of pY15-Cdk1. Wild-type embryos (WT) in interphase of cycle 10 (I10) and cycle 11 (I11) were compared to dwee1 mutants of the same stage.

Figure 2. dwee1 Mutant Embryos Show Spindle and Centrosome Defects

(A–B) Wild-type and dwee1 mutant 1–2 hr embryos were fixed and stained for DNA with Hoechst (blue), α-tubulin (green), and either γ-tubulin (A) or Dgrip84 (B) (red). (A) M12 dwee1 mutants lack astral microtubules in anaphase (compare arrows in [b] and [e]) and form disorganized central spindles in telophase (compare arrows in [c] and [f]). (B) (a and b) Spindle poles from WT but not dwee1 mutant embryos in M13 contain Dgrip84 (arrows). (c and d) dwee1 mutants show spindle poles with many small γ-tubulin foci (arrow in [c]) and broad γ-tubulin-stained poles associated with multiple spindles (arrow in [d]). (C) Analysis of cellularization in the wild-type, dwee1 mutants, and dwee1,mnk double mutants. Embryos 4–19 hr old were fixed and stained with an antibody to phosphorylated tyrosine (PY), which stains cell membranes, and with Hoechst to visualize DNA. Wild-type embryos (WT) show normal nuclear and cellular morphology. dwee1 mutants show no evidence of cell membranes, and DNA appears mostly internalized or in large bodies. In contrast, dwee1,mnk double mutants contain cell membranes and DNA at the embryo cortex. Two representative samples are shown to illustrate the range of phenotypes in double mutants (embryo in [c] exhibits further development and more complete cellularization than embryo in [d]). Pole cells in dwee1 mutants remain at the posterior end of embryos (arrow in [b]), whereas pole cells in some dwee1,mnk double mutants (~60%) can be seen in the process of becoming internalized (arrow in [c]). The scale bar represents 10 μm in (A) and (B) and 50 μm in (C).

Figure 3. Time-Lapse Confocal Analysis of dwee1 Mutant Spindles and Chromosomes

(A) Live wild-type (WT) and (B) dwee1 mutant (dwee1) embryos expressing 17238-GFP were analyzed by time-lapse confocal microscopy. In cycle 13 dwee1 mutant embryos, GFP signal is not detected at spindle poles or on astral microtubules (compare arrowheads in [A] and [B]), and central spindles appear disorganized (compare arrows in [A] and [B]). (C) Live WT and (D) dwee1 mutant embryos expressing histone H2A-GFP (H-GFP) were analyzed by time-lapse confocal microscopy. In dwee1 mutants, anaphase fails in cycle 13, leading to formation of polyploid nuclei (arrows in [D]). Chromosomes in dwee1 mutants attempt anaphase in a much less condensed state than chromosomes in wild-type embryos (arrowheads in [D]). The scale bar represents 10 μm.

Figure 4. Mitotic Defects in dwee1 Mutant Larval Neuroblasts

(A) The percentage of mitotic cells in larval brains from wild-type (WT), dwee1^ES1, or Df(2L)dwee1^wo5 heterozygotes (dwee1/+) and dwee1^ES1 hemizygous mutant larvae (dwee1) was determined. Mitotic index is significantly elevated in dwee1 mutant brains when compared to those of the wild-type (p < .05). (B) A significant percentage of mitotic neuroblasts in dwee1 mutant brains are polyploid (p < .05). (C) Examples of euploid and polyploid neuroblasts from a dwee1 mutant brain. The scale bar represents 5 μm.