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Introduction

Reversible protein phosphorylation and periodic protein destruction play major roles in regulating the eukaryotic cell division cycle. The major protein kinase that directs cell division is cyclin-dependent kinase 1 (Cdk1), the active component of Maturation Promoting Factor, first found to promote meiotic entry in amphibian oocytes. The cyclical inactivation of Cdk1 prior to mitotic exit is brought about in part through destruction of its cyclin partner (reviewed by [1]). Two other protein kinase families, the Polo and Aurora families, are known to have critical functions in progression into and through M phase (mitosis and cytokinesis) and functionally interact with each other and also with Cdk1 to mediate their functions.

Polo, originally discovered in Drosophila [2,3], exemplifies an evolutionarily conserved mitotic protein kinase. Polo, as well as its close orthologs, has been shown to function in multiple events essential for cell division. Polo was initially found to be essential for centrosome maturation and separation [2]. It promotes recruitment of the γ-tubulin ring complex and phosphorylates Asp to facilitate nucleation of an increased number of dynamic microtubules on mitotic entry (reviewed by [4]). At the G2/M transition, Polo (Polo-like kinase 1 in vertebrates) phosphorylates and activates the Cdc25 phosphatase responsible for removing inhibitory phosphates on Cdk1; this promotes mitotic entry [5]. It also functions at the kinetochore-microtubule interface to monitor tension; the 3F3/2 phospho-epitope seen on kinetochores in the absence of tension is a consequence of Plk1/Plx1 kinase activity in vertebrates [6,7]. Removal of cohesins from chromosomal arms in mitosis and meiosis also requires phosphorylation of cohesin subunits by Polo kinases (reviewed by [8]). In Drosophila meiosis II, Polo phosphorylates and inactivates the centromeric cohesion protector protein Mei-S332 [9]. In addition, Polo is required for cytokinesis [10]. The growing list of Polo kinase substrates is evidence of its role in multiple mitotic events.

It is clear that protein kinases such as Cdk1 and Polo are only part of a large network of protein kinases that regulate cell cycle progression, many of which are as yet poorly characterized. A genome-wide survey found that up to one-third of the protein kinome of Drosophila has some cell cycle role [11]. Depletion of the Gwl kinase from S2 cells by RNA interference (RNAi) led to a mitotic delay characterized by formation of long spindles and scattered chromosomes [11]. Yu and colleagues (2004) also found a mitotic role for Gwl kinase by characterizing missense hypomorphic mutants. Reduced gwl function results in mitotic defects in larval neuroblasts and tissue culture cells, including delay between late G2 and anaphase onset and chromosome condensation defects. Gwl has close homologs across eukaryotes and more distant homologs in budding and fission yeasts. Indeed, Yu and colleagues recently reported a function for Xenopus Gwl in mitotic entry, as part of the Cdc2/Cdk1 activation loop in oocyte extracts [12]. In that system, Xenopus Gwl is directly activated by cyclin B-Cdc2 and is in turn needed to promote full activation of cyclin B-Cdc2, although the direct target(s) mediating this action is (are) still unknown and indeed no substrates of Gwl are yet known. The primary sequence of Gwl shows that the regions most homologous to other kinases are split by a long intervening sequence of unknown function [13]. Despite this recent progress, nothing is known about how activity of this crucial kinase is coordinated with the multiple events of cell cycle progression. Moreover, it is not known how Gwl contributes to the different types of mitotic and meiotic cell cycles of a metazoan.

Elucidation of protein function may be aided through the generation of multiple mutant alleles that can reveal separate functions of individual proteins in multiple cellular processes. Drosophila offers the possibility of such studies and, moreover, allows the study of protein function in different types of cell cycle during its development. We applied this principle to study the gene defined by Scant (Scott of the Antarctic), a gain-of-function, dominant enhancer of maternal-effect embryonic defects of polo mutants. Syncytial embryos derived from females heterozygous for both Scant and polo develop mitotic defects in which a centrosome disassociates from one pole [14]. Here we report that the Scant mutation is an allele of gwl that introduces a K97M amino-acid substitution into the Gwl protein; this results in a hyperactive kinase with altered specificity in vitro. Our results indicate an antagonistic relationship between Gwl and Polo and suggest that their activity has to be coordinated for proper embryonic mitotic function. An allelic series of new gwl mutations reveals multiple functions for the Gwl kinase in both mitosis and female meiosis. These display somatic developmental defects accompanied by chromosome condensation and segregation defects in larval neuroblasts. gwl⁺ encodes two isoforms, only one of which is expressed during vitellogenesis. An allele that specifically prevents the expression of this isoform reveals requirements for Gwl in meiosis and in the maternal contribution to the egg.

Results

Scant, a Dominant Enhancer of polo Maternal Effect Lethality, Is an Allele of greatwall

Scant is an ethyl methanesulfonate-induced mutation on the right arm of Chromosome III that causes greatly reduced female fertility when heterozygous with polo¹ (on 3L). These females produce embryos that develop characteristic mitotic abnormalities [14]. Scant shows no interaction with other mutants known to affect progression through embryonic cycles, namely gnu, mgr, asp, and stg [14]. The original Scant-bearing chromosome is also recessive female sterile [14]; however, although they are close, we were able to separate the dominant Scant from the recessive(s) by crossing-over (see Text S1). One recombinant that retained the dominant Scant interaction but was recessive female fertile was recovered; this and its derivatives were used in all the experiments reported here.

Scant is a dominant enhancer of polo female sterility for all polo alleles, though the strength of the interaction varies with the defectiveness of the polo allele. Scant was originally described in combination with polo¹ [14], a hypomorphic allele; polo¹ homozygotes are viable though females are completely sterile. Typically, polo¹ +/+ Scant females produce 4% as many progeny as controls, but polo¹¹ +/+ Scant and Df(3L)rdgC-co2, polo⁻ +/+ Scant females are completely sterile (Figure 1A). polo¹¹ is a breakpoint allele, In(3L)polo¹¹, 77E1–3;77E1–2 (see Text S1), and therefore probably amorphic; homozygotes are prepupal lethal. This suggests that the interaction depends on how much functional maternal Polo protein is available in the egg. However, the reciprocal question—whether the interaction depends on how much functional Scant protein is in the egg—cannot be asked; Scant is not only homozygous viable and fertile, it is viable and fertile over all deficiencies of its region, and none of these deficiencies affect fertility of heterozygous polo mutations. This suggests that the Scant mutation is hypermorphic or neomorphic. If it is, then the dominant interaction with polo mutations should be alleviated by mutations that inactivate its gene, and those mutations may have recessive phenotypes that can be deficiency mapped. We therefore recovered Scant revertants by mutagenizing homozygous males with X rays, mating them to polo¹¹/Balancer females, and testing the + Scant*/polo¹¹ + daughters for fertility. We recovered three classes of revertants (Figure 1B):

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.

doi:10.1371/journal.pgen.0030200.g001

The first class is duplications of polo⁺: we recovered two, a tandem duplication and a 3;3 duplication transposition. This confirms the deduction from the genetic analysis that, in the presence of the hyper- or neomorphic Scant mutation, two maternal doses of polo⁺ are needed for full female fertility.

The second class is a second-site suppressor because it is a 3L deficiency and independent overlapping deficiencies of its region also suppress the polo–Scant interaction; we have yet to identify the relevant gene.

The third class is represented by two recessive mutants in this first mutagenesis; they fail to complement each other genetically and were named Scant revertants (Sr3 and Sr6; see Text S1). The chromosome carrying the second-site suppressor complements the recessive phenotypes of the third class.

Sr3 is homozygous viable with eclosion delay; the adults have variably rough eyes, eroded wing margins, and missing bristles, all symptoms of mitotic defects, and both sexes are sterile with strongly reduced gonads. There are few germ cells even in newly eclosed adults; none remain a few days later. Sr6 homozygotes die as pupae but this reflects a second lethal on its chromosome; Sr3/Sr6 adults eclose, with the above constellation of phenotypes, though viability is reduced. Viability of Sr3 and Sr6 is further reduced over noncomplementing deficiencies, so the allelic series is Sr3 (hypomorph), Sr6 (more defective hypomorph), deficiency (see below). We sequentially uncovered the two recessive Sr alleles by three deficiencies: the first extends from 91A8 to 91F5; the second from 91B4 to 91D3; and finally between genome sites 14566000 and 14750000 (Df(3R)Exel6181, 91C5;91D5). This restricts Scant to one of 16 potential genes in an interval of 185 kb.

To further refine the Sr mapping, we mobilized four different viable P-element insertions in or near this 185-kb region to generate imprecise excisions (Figure 1C; Text S1) with delta 2–3 transposase. All progeny receiving the hopping P-element chromosome were crossed either to Df(3R)Exel6181 or Sr3. This allowed us to determine whether a lethal excision had occurred or whether an excision or hop failed to complement the Scant revertant. One particular line, gwl ^EP515, whose P element is inserted 217 bp 5′ of gwl and 12 bp 5′ of CG7718, yielded several hits (Figure 1C). Complementation tests define three groups of mutations: (1) three independent events (gwl², gwl^3a, and gwl^6a) that fail to complement Sr3; (2) three independent events (CG7718¹, CG7718^6b, and CG7718⁷) that complement Sr3 but fail to complement each other; and (3), one event (gwl^3b) that fails to complement mutations of both groups. PCR revealed that the gwl gene is disrupted in mutations of the first group from imprecise excision extending in one direction. CG7718 is disrupted in the second group from imprecise excision extending in the other direction. Finally, both gwl and CG7718 are disrupted in the third event; the imprecise excision extends in both directions. The failure of gwl², gwl^3a, and gwl^6a to complement Scant revertant alleles suggests that the Scant mutation affects the gwl gene; this was confirmed by sequencing gwl in them (see “Scant Encodes a Hyperactive Gwl Kinase with Altered Specificity” and “The Allelic Series of Recessive gwl Alleles Reveals Multiple Mitotic Functions” below). gwl² and gwl^3a are probably deletions into gwl, and gwl^6a clearly is; both 5′ upstream sequence and N-terminal coding sequence have been deleted. All three of these small deletions give escapers homozygous and over deficiencies on nutritious food, with the Sr constellation of phenotypes. Thus, the gwl gene is not absolutely vital.

Finally, we carried out a second X-ray mutagenesis, now of a polo¹¹ Scant recombinant chromosome made from the fertile polo¹¹/Dp(polo⁺) Scant females and tested over a third-chromosome balancer (Figure S1). This gave ten positives, all of class three; four are cytologically visible deficiencies each of which removes all or part of 91C5–6 (plus more), one is a translocation with one breakpoint in 91C, and the remaining five have no obvious relevant cytological defect. For two of these, all or part of the gwl gene fail to PCR up, so they are probably additional deletions. All seven of these fail to complement Sr3 for both phenotype and viability. The final three, Sr17, Sr19, and Sr18, complement Sr3 and deficiencies for phenotype but females have reduced fertility or are sterile. Sr17 and Sr19 also have reduced viability over Sr3 in the presence of polo¹¹ and some DNA defect 5′ of the gwl coding region (probably an insertion based on PCR, see Text S1). Both alleles reduce the amount of Gwl protein produced but have not been studied further. Sr18 is perfectly viable but is absolutely maternal-effect female sterile; this allows us to separate somatic gwl function from its role in producing a functional egg. All further studies of Sr18 used a chromosome from which polo¹¹ had been removed by recombination.

Thus we identified three categories of gwl alleles: the dominant Scant allele, recessive zygotic plus germline alleles, and a recessive maternal-effect allele. Our characterization of these three groups gives new insights into the mitotic and meiotic functions of the protein kinase that gwl encodes.

Scant Enhances polo Defects at Spindle Poles

The severity of the dominant Scant mutant phenotype increases in relation to the decrease in polo function. Not only is the dominant effect overcome by two doses of polo⁺ in the presence of a polo mutation (see above), but also the severity of the phenotype in the presence of one copy of polo⁺ is proportional to the strength of the polo allele. Weak hypomorphic alleles such as polo¹, a viable allele that shows maternal effect lethality, give some progeny when heterozygous with Scant. Amorphic alleles such as polo¹¹ (see above) and polo⁻ deficiencies are completely sterile; these females lay normal numbers of eggs that do not hatch but do begin to develop and turn brown.

The archetypal Scant phenotype is shown by embryos derived from polo^mut +/+ Scant females. The mitotic figures of such embryos frequently display centrosome disassociation from one pole (Figure 2A and 2B). To compare severity of phenotypes we counted the number of defective mitotic nuclei (showing detachment of at least one centrosome) in syncytial embryos derived from mothers of different genotypes (Figure 2B and 2C). There is a slight but significant increase in defective spindles in embryos derived from polo¹/+, polo¹¹/+, Scant/+, and Scant/Scant females relative to wild-type females, indicating that a single mutant copy of these genes in mothers leads to mitotic defects at a low frequency (Figure S2). However, these embryos always hatched and developed fully, indicating that such low frequencies of defects can be tolerated. The mitotic spindles in embryos derived from polo¹¹ +/+ Scant females are more frequently aberrant than those derived from polo¹ +/+ Scant females. Thus the severity of the maternal effect phenotypes observed at the cellular level is consistent with the relative strengths of the polo alleles as homozygotes, indicating that, in the context of the early embryo, Scant enhances mitotic defects resulting from a decrease in polo function.

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).

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To examine the formation of the aberrant mitotic figures in polo¹¹ Scant-derived embryos, we used time-lapse microscopy to follow mitosis in embryos that also expressed green fluorescent protein (GFP)-β-tubulin constitutively from the ubiquitin promotor (Figure 3; Videos S1 and S2). No defects were observed in embryos derived from the wild-type GFP-β-tubulin stock (Video S1). In contrast, polo¹¹ Scant/+ +-derived embryos show an initial detachment of one centrosome early in mitosis, before nuclear envelope breakdown. All 33 cases where the filming was continuous from before the centrosome detached show that the detachment occurs prior to nuclear envelope breakdown and involves only one centrosome. The free centrosome drifts away from the nucleus, and astral microtubule formation usually appears normal, though there is no asymmetric microtubule enhancement. A half-spindle is established by microtubules forming connections between the chromosomes and the centrosome still associated with the nuclear envelope. However, spindle bipolarity is often attained by microtubules growing from the chromosomes outwards. If a free centrosome is sufficiently close to this second half-spindle, it can reattach to it to form a normal bipolar spindle containing two centrosomes and nuclear division completes normally (Figure 3A; Video S2). However, if the free centrosome drifts too far away from its spindle, it cannot be recaptured, and the monoastral spindle that forms initially is unfocussed at the pole lacking a centrosome. In some cases, monoastral bipolar spindles fuse with neighboring spindles (Figure 3B; Video S2) and degenerate to give interconnecting arrays of microtubules (Figure 2A). In other cases, the acentrosomal pole eventually focuses and anaphase occurs (Video S2) (unpublished data) as previously observed in sak mutants, which lack centrosomes [15]. The primary defect in these mitoses is therefore premature centrosome detachment; subsequent spindle abnormalities reflect secondary, mechanical problems. Somehow the Polo-Gwl kinase balance is important for maintaining centrosome-nuclear envelope propinquity until nuclear envelope breakdown.

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.

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Free centrosomes always show the presence of γ-tubulin, pericentrin-like protein (PLP) and centrosomin (CNN) when the attached one does, while the acentrosomal spindle poles always lack all three antigens (unpublished data)—free centrosomes seem to mature normally. Indeed, free centrosomes are mature by the functional test as well, since they can serve as active poles if they become reassociated with a spindle (Figure 3A).

Loss of centrosomes in Drosophila embryos has been shown to occur in response to DNA damage; nuclei then drop from the cortex into the interior of the syncytial embryo [16]. Both centrosome loss and nuclear fallout are suppressed in embryos lacking the Chk2 protein kinase [17]. Centrosomes still detach in embryos derived from chk2/chk2; polo¹¹ Scant/+ + females (Figure S3), suggesting that the loss of centrosomes in embryos laid by polo¹¹ Scant females is not the consequence of DNA damage inducing its response pathway. This independence from Chk2 as well as the enhancement of polo phenotypes by the Scant mutation suggests that coordinated activity of the protein kinases encoded by these genes is required directly to coordinate centrosome attachment to the nuclear envelope before spindle assembly.

Scant Encodes a Hyperactive Gwl Kinase with Altered Specificity

To identify the nature of the Scant mutation, we sequenced gwl on both the Scant chromosome and on several non-Scant mutant chromosomes that resulted from the same mutagenesis [14]. The sole difference is an A to T base change in Scant, which changes amino acid residue 97 from lysine to methionine. The gwl sequence in the Sr mutations retains the K97M substitution codon as well as additional changes, consistent with their recessive reduction in function (see “The Allelic Series of Recessive gwl Alleles Reveals Multiple Mitotic Functions” below).

To confirm that the K97M substitution in Gwl is indeed responsible for the Scant phenotype, we reconstituted the genetic interaction with polo using a synthetic gwl-K97M generated in a wild-type gwl sequence and carried as a transgene. Because the Scant mutation appears to be hyper- or neomorphic, we also asked whether expressing wild-type (wt) Gwl kinase at higher levels than normal generates the characteristic embryonic mitotic defects from polo/+ mothers. There are two isoforms of Gwl; the female vitellogenic ovary expresses only the long form (see “The Long Isoform of Gwl Is Required for Female Meiosis and Is Provided to the Egg” below). We therefore made transgenic flies expressing either the longer isoform of Gwl-Wt or Gwl-K97M (otherwise identical to wild type) under the control of the UASp promotor, driven by Gal4 expressed from the maternal α-tubulin promotor (Mat α-Tub Gal4). Expression of the transgenes (checked by western blot; unpublished data) was driven at comparable levels in the germline in polo¹¹/polo⁺ and polo⁺/polo⁺ females, and in both cases the transgenic expression exceeds the endogenous Gwl by approximately 3-fold (unpublished data). Female germline expression of Gwl-K97M in polo¹¹/polo⁺ heterozygotes causes complete sterility (Figure 4A). These embryos show loss of centrosomes from very early mitotic spindles (Figure 4BIV), a much stronger and earlier phenotype than Scant itself. Overexpression of Gwl-wt in heterozygous polo¹¹/polo⁺ females allows partial egg hatch (around 4%), and these embryos also show loss of centrosomes from spindles in early syncytial divisions (Figure 4BIII). Fertility is also reduced (to 23%) in polo⁺/polo⁺ females expressing Gwl-K97M where centrosome loss usually occurs in the later divisions of the syncytial blastoderm stage (Figure 4BII). However, polo⁺/polo⁺ females overexpressing Gwl-wt are fully fertile and their embryos do not show significant centrosome detachment (Figure 4BI). Thus embryonic mitotic defects and maternal-effect lethality arise from overexpression of either the wild-type or K97M mutant forms of Gwl kinase in the female germline when Polo kinase function is reduced, but only the K97M form induces a phenotype when Polo function is normal.

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.

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The above results show that the Scant phenotype in embryos with compromised Polo function can arise just from increased maternal expression of Gwl kinase but also indicate that the Gwl-K97M protein has additional properties, perhaps increased stability or altered activity. Western blots of gwl⁺ and Scant-derived embryos show no increase in levels of Gwl resulting from the Scant mutation; if anything there is less Gwl in homozygous Scant, so an increase in stability is unlikely (Figure 4C). To ask whether Scant's K97M mutation affects the kinase activity of Gwl, we expressed Myc-tagged forms of Gwl (WT or K97M or kinase-dead [K87R]) in Drosophila cells in culture and purified the fusion proteins for in vitro kinase assays (Figure 4D). Myc-Gwl phosphorylates myelin basic protein and casein more efficiently than histones H1 or H3, although the signals are only marginally increased above the K87R, presumably kinase-dead, control (the K87R mutation may not effectively abolish the kinase activity or phosphorylation could result from traces of copurifying kinases). Myc-Gwl-K97M specifically phosphorylates myelin basic protein with dramatically increased efficiency, while showing only slight increases for casein and H1 and no increase for H3. This high activity on myelin basic protein was repeatedly observed despite the lower amount of kinase present in the reaction for Myc-Gwl-K97M compared with Myc-Gwl or Myc-Gwl-KD. Indeed we found that Gwl-K97M was reproducibly expressed at a lower level than its WT equivalent in several independent stable cell lines. This suggests that the hyperactive K97M mutation is toxic to the cell line used, so the only stable transformants that survive are those that can keep it downregulated. Both our genetic and biochemical results show that the Scant mutation (Gwl-K97M) results in a hyperactive enzyme with altered specificity.

Taken together, several pieces of evidence indicate that Gwl and Polo have antagonistic activities in the early embryo. The Scant phenotype is not seen in the presence of wild-type levels of polo, and its strength is dependent upon the strength of the mutant polo allele. The failure of embryos to develop upon overexpression of overactive Gwl-K97M and, to a lesser extent, of Gwl-wt, is strongly dependent on reduced Polo dosage. Two of the mutations that restore fertility to polo-Scant heterozygous females are duplications of polo⁺ (Figure 1B; Text S1). These results suggest negative regulatory interactions between Gwl and Polo and indicate that the balance between these two protein kinase activities is paramount for success of the rapid mitotic cycles of early embryonic development.

The Allelic Series of Recessive gwl Alleles Reveals Multiple Mitotic Functions

The hypomorphic recessive alleles of gwl allowed us to study the roles of the Gwl protein kinase in several of the different cell cycle types in Drosophila development. We compared their mutant phenotypes to those of the null allele gwl^6a, a P-hop deletion that removes the N-terminal end of the Gwl protein and its 5′ upstream sequence (described above). Two close bands are detectable around 100 kDa in Gwl western blots from larval brains, and both bands are absent in hemizygous gwl^6a larval brains (Figure 5B).

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.

doi:10.1371/journal.pgen.0030200.g005

DNA sequencing revealed that all of the nondeficiency Scant revertant alleles (Sr) retained the K97M Scant mutation (Figure 5A). gwl^Sr3 has, in addition, a 27-bp deletion corresponding to amino acids 156–164 (Figure 5A). This short deletion maps to the predicted kinase fold in the N-terminal portion (not shown). The gwl^Sr3 allele is a hypomorph since gwl^Sr3/gwl^Sr3 is more viable than gwl^Sr3/Df; and gwl^Sr3/Df is in turn more viable than gwl^null/Df. This predicts that the kinase encoded by the gwl^Sr3 allele retains some activity. However, western blots of extracts of gwl^Sr3/Df larval brains reveal little or no protein, suggesting that the protein encoded by gwl^Sr3 is unstable (Figure 5B). gwl^Sr6 has an A to T substitution that changes lysine 689 into the ochre termination codon (STOP; Figure 5A). gwl^Sr6 also behaves as a hypomorphic allele suggesting either that this STOP codon can be suppressed to some extent or that the truncated protein retains partial function. We see no Gwl protein in western blots of gwl^Sr6/Df larval brain extracts (Figure 5B) but our anti-Gwl antibodies were raised against the C-terminal part of the protein that is predicted to be truncated in this mutant, so this test is unreliable. The weak hypomorphs gwl^Sr17 and gwl^Sr19 show a slight reduction in levels of both forms of Gwl in brains (unpublished data). Finally, the female-sterile-only allele, gwl^Sr18, has one base deleted in the splice acceptor sequence of exon 4. This change in sequence from AAAGGGCT to AAAGGCT (Figure 5A) leaves the same splice-acceptor sequence but changes the reading frame after it to encode a string of 61 different amino acids followed by a series of STOP codons. This mutation has lost the slower migrating isoform of Gwl (Gwl-long) in neuroblasts of gwl^Sr18/Df larvae without affecting the faster form (Gwl-short) (Figure 5B). Both isoforms of Gwl are also expressed in S2 cells (Figure S4) [13]. We cloned both Gwl cDNAs from total S2 cell mRNA; sequencing revealed that the shorter cDNA lacks exon 4 precisely. RNAi treatment of S2 cells using double-stranded (ds)RNA targeting exon 4 depleted the upper but not the lower Gwl band seen on western blots (Figure S4), whereas RNAi to common exons depleted both isoforms. Thus gwl^Sr18 has a mutation in a splice site used in the synthesis of only one of the two isoforms.

The mutations fall into an allelic series. The amorphic genotype gwl^6a/Df dies predominantly in the pupal stage, and the rare escaper adults have rough eyes, ragged wings, cuticle defects, missing bristles, and are sterile, phenotypes typical of cell division cycle mutants. We found similar, but less severe, phenotypes in gwl^Sr3/Df, gwl^Sr6/Df, and gwl^Sr3/gwl^Sr6 animals, a greater proportion of which survive to adulthood (Figure S5). In contrast, gwl^Sr18/Df flies are fully viable and show no such morphological defects but females are sterile (males are fertile).

The severity of developmental defects is paralleled by the cellular phenotype of third instar larval neuroblasts in the gwl mutants (Figures 6 and S6). Strikingly, the viable but female-sterile splice acceptor site mutant that expresses no long isoform, gwl^Sr18, shows no significant mitotic defects. The other Scant revertant alleles (illustrated here by gwl^Sr3/gwl^Sr6) all show mitotic defects that are exaggerated in hemizygotes, and gwl^Sr6 is usually more affected than gwl^Sr3 (Figure S6). In Figure 6, the strongest phenotypes are seen in gwl^6a/Df. There is an increase in the mitotic index; more cells are pre-anaphase (Figure 6B). RNAi targeted to the first exon of gwl in cultured S2 cells depletes both isoforms and also results in an increase in mitotic cells, confirming earlier findings [11]. These cells show BubR1 staining on kinetochores and high cyclin B levels (Figure S4), indicating that they are delayed in prometaphase. gwl mutant neuroblasts also show a high frequency of defective chromatin condensation. However, in contrast to the undercondensation reported in other gwl alleles by Yu and colleagues [13], we consistently observe that some regions of chromosomes appear undercondensed and other regions are overcondensed (Figure 6A). In some cells the chromosomes are uniformly overcondensed, suggesting lengthy pre-anaphase delay (Figure 6A). In S2 cells depleted of Gwl by RNAi, we do not observe strong undercondensation, but rather chromosomes are scattered along extensively elongated spindles (Figure S4), as also found by Bettencourt-Dias et al. [11]. The small number of cells that manage to enter anaphase in mutant neuroblasts frequently show anaphase bridges (Figure 6A). The gwl null gwl^6a/Df, unlike the hypomorphs, has a significant proportion of polyploid mitotic figures (Figure 6A and 6B). Conditions that block anaphase, such as colchicine treatment, also induce polyploidy; eventually the hypercondensed metaphase chromosomes decondense, and the cell reenters interphase, bypassing the mitotic checkpoints. Since gwl^6a/Df cells show significant pre-anaphase delay with hypercondensed chromosomes, polyploidy is in fact expected from this normal interphase reentry. The absence of polyploid cells in the hypomorph gwl^Sr3/gwl^Sr6 suggests that enough active Gwl is available to complete mitosis normally—eventually.

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.

doi:10.1371/journal.pgen.0030200.g006

Expression of UASp-gwl-long or UASp-gwl-short as transgenes driven ubiquitously by Actin 5C-Gal4 rescues the viability and somatic integrity of gwl^Sr3/gwl^Sr6, gwl^Sr3/gwl^Sr3, gwl^Sr3/Df, and gwl^Sr6/Df flies (Figures 6C and S5) (unpublished data) to similar degrees. Therefore, both forms of Gwl (long and short) are active kinases that have redundant functions in somatic tissues where they are both present (Figure 5B).

The Long Isoform of Gwl Is Required for Female Meiosis and Is Provided to the Egg

gwl^Sr3, gwl^Sr6, and gwl^6a, in which levels of Gwl are reduced (at least in neuroblasts; Figure 5B), are all sterile in both males and females. This sterility reflects cell-proliferation failure of the germlines and probably of their supporting somatic tissues as well. In contrast, gwl^Sr18/Df shows normal viability and has no somatic defects including normal ovaries and testes, despite expressing only the short isoform of Gwl (Figure 5B). Therefore, the short form is sufficient for mitosis in general, and it is both necessary and sufficient for the mitotic divisions of germline cells in ovaries and testes. gwl^Sr18/Df females lay lots of eggs but they remain white; males are fertile. Since gwl^Sr18 produces eggs, the Gwl-long isoform has a specific role late, rather than early, in germline function. Western blotting of mature wild-type ovaries or unfertilized eggs does show high levels of Gwl (Figure 7A and 7B); a single band in unfertilized eggs (Figure 7B) and a thick band in ovaries (Figure 7A). In contrast, gwl^Sr18/Df females show a great reduction in Gwl signal in these tissues. Since our molecular analysis (cloning and sequencing) and biochemical analysis (in neuroblasts) reveal that only the long form is affected by the gwl^Sr18 mutation and since Gwl signal disappears from gwl^Sr18/Df eggs and ovaries, the western signal in wild-type eggs and ovaries arises from the long form only. Gwl-short was not detected in eggs or mature ovaries, although Gwl-short must be present in gwl^Sr18/Df ovarian follicle cells and premeiotic germline mitotic cells, since these do divide normally. Gwl-long appears as a thick band in western blots of wild-type ovary extracts (Figure 7A); this presumably corresponds to activated phosphoforms of Gwl-long analogous to those seen in [12], while it appears as a thin band in blots of unfertilized eggs (Figure 7B), where Gwl-long may not be activated.

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.

doi:10.1371/journal.pgen.0030200.g007

When we examined the ovaries of gwl^Sr18/Df females we found them fully developed (Figure 7C). However, preliminary observations reveal that late (stages 13–14) eggs often have irregularly distributed yolk, in no fixed pattern. Earlier stages have uniform yolk distribution but appear to have less of it than same-stage controls (our unpublished observations). The Drosophila ovary comprises 15 or so ovarioles each containing multiple egg chambers at successive stages of development. Cystoblasts in the germaria of the ovarioles divide four times to form cysts of 16 cells that remain interconnected by ring canals derived from the cleavage furrows of incomplete cytokinesis. Of the two cells interconnected by four ring canals, one will become the oocyte although formation of synaptonemal complex is complete in both of them. Once the oocyte is determined, the other 15 cells undergo endoreduplication cycles and associated cell growth to become the nurse cells. The morphology of egg chambers of gwl^Sr18/Df females appears normal; they have 15 nurse cells and one oocyte indicating that the preceding mitoses had progressed normally. We detect strong staining for Gwl (the antibody recognizes both isoforms) in wild-type egg chambers from stage 8 of oogenesis onwards but not in gwl^Sr18/Df females (the faint signal in gwl^Sr18/Df ovaries is probably Gwl-short present in the follicle cells surrounding the egg chamber but some may be background). In wild-type egg chambers, Gwl protein is present in the oocyte and four neighboring nurse cells; actin staining shows that all four nurse cells connect directly to the oocyte by ring canals and that they have the expected numbers of ring canals to be the oocyte's sister and daughters (four, three, two, and one; unpublished data). In each of these cells Gwl accumulates in the nucleus, although there is some cytoplasmic staining. The long isoform of Gwl is contributed maternally to the embryo where it also concentrates in interphase syncytial nuclei. During mitosis Gwl is depleted in the nucleus and enriched around the outside of the spindle envelope before accumulating in the nucleus once again during the next interphase (Figure S7). Thus Gwl-long accumulates in a subset of polyploid nuclei of nurse cells, the prophase I nucleus of the oocyte and interphase nuclei of the embryo.

Gwl accumulation in the oocyte led us to ask whether gwl^Sr18/Df oocytes encounter problems during meiosis per se. To examine female meiosis I, we performed immunostaining (for α-tubulin and DNA) in inactivated vitellarial eggs at stages 13 and 14. Wild-type oocytes normally arrest in metaphase I with the larger chromosomes with chiasmata compacted into a single mass at the metaphase plate, while the small fourth chromosomes have moved halfway to the poles as the result of distributive segregation (see WT control in Figure 8Aa and [18,19]). We scored the number of DNA masses (ignoring the tiny fourth chromosome) in WT and gwl^Sr18/Df oocytes. In contrast to the WT oocytes, which mostly have only one DNA mass at the metaphase plate, 78% of gwl^Sr18/Df oocytes have widely scattered chromosomes, and the number of chromosome masses varies from two to 12 (Figure 8B). Complete homologue separation would give six large chromatin masses; about a quarter of the mutant meioses have more masses than that. Multiple chromatin masses might be caused by reduced meiotic exchange, or precocious loss of sister-chromatid cohesion, or broken chromosomes resulting possibly from faulty meiotic exchange. If meiosis I lacks chiasmata then metaphase I arrest does not occur [18,19], although in wild type anaphase I and II proceed precociously but normally; although some anaphase movements probably occur precociously in gwl^Sr18/Df, they are certainly not normal, so simple reduced exchange does not explain this phenotype though it could be compounded with other problems. A few of the meiotic spindles are aberrantly shaped (Figure 8C; 8% of the total oocytes), but all of the multipolar spindles contain multiple DNA masses. Since the wild-type spindle is nucleated from the central mass of chromatin in meiosis I, these abnormal spindles probably reflect nucleation of individual “mini-spindles” from the multiple chromosome masses.

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.