Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase

Abstract

Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. We show that Scant creates a hyperactive form of Greatwall (Gwl) with altered specificity in vitro, another protein kinase recently implicated in mitotic entry in Drosophila and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo-Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. Our results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle.

Figure 1. Generation of gwl Alleles

(A) Females heterozygous for both the Scant mutation and a loss-of-function polo mutation (such as polo¹¹) lay embryos that die during development. (B) Genetic screen to generate Scant revertant (Sr) alleles. Males homozygous for Scant were x-rayed and crossed to polo¹¹ heterozygous females. Female progeny were tested for fertility. Scant revertant mutations restore female fertility and can be duplications of the polo⁺ gene, third-site suppressor mutations (su), or recessive mutations allelic to each other. See Text S1 for details. (C) Hopping a P-element inserted directly upstream of the gwl gene generated imprecise excisions disrupting either gwl (gwl², gwl^3a, and gwl^6a), CG7718 (CG7718¹, CG7718^6b, and CG7718⁷) or both genes (gwl^3b). gwl², gwl^3a, and gwl^6a are semilethal and produce a “Scant-revertant (Sr) phenotype” when placed over Sr alleles (generated in [B]). The extent of the imprecise excisions was approximately mapped by the ability to PCR-amplify the 1 kb-long regions of genomic DNA defined by the tick marks.

Figure 2. Scant Interacts Genetically with polo, Leading to Mitotic Defects in Embryos

(A) Aberrant mitotic figures in embryos derived from females heterozygous for polo and Scant. Embryos were collected for 2–3 h, dechorionated, and fixed for immunofluorescence. Stainings are α-tubulin (green), γ-tubulin (red), and DNA (blue). Representative examples of mitotic phenotypes are shown for the indicated genotypes. Arrows in the center panel indicate the displacement of centrosomes from one pole. Scale bars are in μm. (B) Typical displacement of one centrosome observed in embryos derived from polo¹ +/+ Scant heterozygous females. The scale bar represents 10 μm. (C) Quantitation of aberrant mitotic figures observed. Embryos were treated as in (A) and syncytial single embryos with mitotic nuclei were scored for the percentage of defective nuclei (having lost at least one centrosome). Numbers are average percentages (± standard error of the mean).

Figure 3. Centrosome Dissociation Is Observed in Prophase in polo-Scant-Derived Embryos

Time-lapse fluorescence microscopy of embryos derived from GFP-β-tubulin; polo¹¹ Scant/+ + mothers. In both (A) and (B), centrosomes dissociate prior to nuclear envelope breakdown. (A) In some cases, the disassociated centrosome (arrow) can be recaptured and mitosis can proceed normally. (B) In other cases, centrosomes are lost irreversibly and gross mitotic defects develop. Both series (A and B) were taken from Video S2. Scale bars correspond to 10 μm.

Figure 4. Gwl-K97M Is Hyperactive

(A) Effects of overexpressing gwl-long-wt or gwl-long-K97M (Scant) on female fertility. Mat α-Tub-Gal4 and the UASp-gwl transgenes were present in only one copy on the same chromosome in all flies tested. Single females of the indicated genotypes with 3 WT (Oregon R) males laid eggs for 3 consecutive d. Numbers are averages of hatched adult progeny (± standard error of the mean) per day for 12 females of each genotype (three females of each of four independent transgenic lines per genotype = 36 samples). The 100% reference comes from the observation of 100% egg hatch and no larval or pupal lethality. (B) Effect of overexpressing gwl-long-wt or -K97M (Scant) on embryonic mitosis. Embryos were laid by mothers of the indicated genotypes and treated as in Figure 2A. Scale bars are in μm. (C) Endogenous Scant protein is not more abundant than Gwl-wt in embryos. Western blots for Gwl and Polo from embryos from mothers of the indicated genotypes. *, cross-reactive band that serves as a loading control. (D) Gwl-K97M is hyperactive in vitro with altered specificity. Myc-tagged forms of Gwl-wt, K87R (kinase dead), and K97M (Scant) were expressed in Dmel stable cell lines. Myc immunoprecipitations were carried out and the kinase activity on Histones H1, H3, Casein, and Myelin Basic Protein was assayed on beads. Note that the Gwl-K97M was always expressed at lower levels in stable cell lines, suggesting toxicity for this protein.

Figure 5. gwl Alleles Generated and Used in This Study

(A) Molecular map of gwl alleles. Gwl's highly conserved kinase domain (white) is predicted to be split by a less-conserved intervening sequence of yet unknown function (light gray). The N-terminal and C-terminal ends are also less conserved (dark gray). This diagram is inspired from the one proposed in Yu et al. [13]. The Scant mutation changes lysine residue 97 to methionine, making Gwl hyperactive and altering its specificity in vitro. Mutation Scant revertant 3 (gwl^Sr3) removes exactly nine codons coding for residues 156–164 predicted to be part of the kinase fold. Mutation Scant revertant 6 (gwl^Sr6) introduces a premature ochre termination codon instead of lysine 689. Genetically, both mutations gwl^Sr3 and gwl^Sr6 seem to have residual Gwl function. Mutation gwl^Sr18 changes the reading frame following the splice acceptor site at the beginning of exon 4, which encodes residues 382–424 in Gwl-long. Exon 4 is spliced out from Gwl-short and, therefore, Gwl-short is unaffected by the gwl^Sr18 mutation. Mutations gwl², gwl^3a, and gwl^6a are null alleles generated by imprecise excisions of a P-element (see Figure 1C). (B) Anti-Gwl western blot on larval brain extracts of the indicated genotypes. Gwl-long and Gwl-short are both visible in the WT lane (+/Df). Two gwl^Sr18/Df samples were loaded to confirm that the absence of the top Gwl band is not artifactual.

Figure 6. Gwl Is Required for Normal Mitosis in Somatic Tissues

(A) Chromosomal defects and polyploidy observed in gwl mutant larval neuroblasts following orcein DNA staining. Control prometaphase (a) and anaphase (b) figures are shown, with normal condensation. Examples of undercondensation (c), overcondensation (d), anaphase bridges (e and f) and polyploidy (g and h) observed in gwl mutants are shown. The scale bar is for all images and shows 10 μm. (B) Quantitation of defects observed in larval neuroblasts for WT (control), gwl^Sr18 (female germline specific), gwl^Sr3/gwl^Sr6 (partial loss of function), and gwl^6a/Df (complete loss of function). Mitotic cells were recognized by their condensed chromatin. The M/A ratio corresponds to the number of prometaphase and metaphase cells over the number of anaphase and telophase cells (F = number of fields scored). % condensation defects, number of mitotic cells with under- or overcondensation/total number of mitotic cells (n); % chromatin bridging, number of bridged anaphases/total number of anaphases (n); % polyploidy, number of polyploid mitotic cells/total number of mitotic cells (n). (C) Rescue of gwl^Sr3/gwl^Sr6 mutants by the expression of UASp-gwl-long or UASp-gwl-short from the ubiquitous Actin5C-Gal4 driver. The percentages of expected adult progeny of the indicated genotypes if the viability was normal is shown (numbers at the end of each bar indicated the total number of internal reference progeny flies scored). See Text S1 for details.

Figure 7. Gwl Is Supplied by the Mother to the Developing Egg

(A) Anti-Gwl western blot from ovaries of gwl^Sr18/Df or WT (Oregon R) females. (B) Anti-Gwl western blot from extracts of eggs laid by gwl^Sr18/Df or WT (Oregon R) virgin females. Total proteins were stained by amido black, shown as a loading control. *, cross-reactive band acting as a loading control. (C) Localization of Gwl in ovaries. In addition to Gwl (green), lamin (red) was also stained to show the outlines of the cells and of their nuclei. In WT ovaries, Gwl is clearly visible in the nucleus and the cytoplasm of the oocyte and in the nuclei and cytoplasm of the four nurse cells connected to it. In gwl^Sr18/Df ovaries, only follicle-cell staining of Gwl-short and/or background staining is detected. Scale bars are in μm.

Figure 8. Gwl Is Required for Female Meiosis

(A) Examples of meiotic defects observed in gwl^Sr18/Df oocytes. Alpha-tubulin is stained green and DNA is red. Note the small, probably nondisjoined Chromosomes 4 in b-b'. In c', arrows indicate likely separated sister chromatids of the fourth chromosome. A typical wild-type (Oregon R) meiotic figure arrested in metaphase I is shown for comparison (a). The longer spindles in b and c are a typical feature of the bipolar spindles formed in gwl^Sr18/Df oocytes. Scale bar is 10 μm. (B) Quantitation of chromosomal defects observed in metaphase and anaphase oocytes. In wild-type oocytes, only 2% appear to have progressed into a normal anaphase (not included in this quantitation). Chromosome defects were characterized by scattered chromosome masses. The number of DNA masses, excluding the tiny fourth chromosome, was counted in defective figures. The percentage of defective figures presenting two to six DNA masses (as in Ab–c) or seven to 12 DNA masses (as in Ad–e) is shown. Normal figures showed either one mass in metaphase or two masses in anaphase. (C) Quantitation of spindle defects observed in metaphase and anaphase oocytes. Defective, multipolar, and occasionally monopolar spindles are observed in gwl^Sr18/Df oocytes (as in Ad–e). 149 Oregon R oocytes and 142 gwl^Sr18/Df oocytes were scored in these experiments (B and C). (D and E) Rescue of gwl^Sr18/Df female fertility with the expression of UASp-gwl-long (D) or UASp-gwl-short (E) driven from the Mat α-Tub-Gal4 driver (in two separate experiments). The average numbers of adult progeny per female from 6 d (D) or 7 d (E) of egg laying by females of the indicated genotypes are shown (± standard error of the mean). See Text S1 for details. Females of the genotypes used in (E) were also tested for meiotic chromosome nondisjunction by crossing to Y^SX^.Y^L, v f B/0; C(4)RM, ci ey^R/0 males. Numbers of (nullo-4, diplo-X+nullo-X)/total progeny are, from top to bottom: (0, 0)/432; (2, 0)/671; no progeny; (0, 0)/646; (0, 0)/397; and (2, 0)/294.

Figure 9. Gwl Is Required for Sister Chromatid Cohesion in Female Meiosis

(A) Confocal images of meiotic figures of single oocytes stained for DNA (red) and showing FISH signal for satellite DNA near the centromere of the X chromosome (large green foci). In wild-type oocytes (a–c), one or two large foci are observed, representing the two homologous pairs of centromeres without tension in prophase (one focus) or under tension on the chiasmate bivalent in prometaphase or metaphase I (two foci). Up to four large foci are observed in gwl^Sr18/Df oocytes (d–h), indicating the absence of sister chromatid cohesion. Panels d and e show examples of figures with six or fewer DNA masses; panels f–h show examples of figures with more than six DNA masses. Scale bar is 10 μm. (B) Quantitation of the number of centromeric FISH foci relative to the number of DNA masses in wild-type and gwl^Sr18/Df oocytes (as in [A]). In gwl^Sr18/Df oocytes, the presence of more than two centromeric FISH signals strongly correlates with the presence of more than six large DNA masses, consistent with the loss of sister chromatid cohesion. Numbers of oocytes scored are 75 for wild type, 98 for gwl^Sr18/Df with six or fewer DNA masses, and 32 for gwl^Sr18/Df with seven or more DNA masses.