Drosophila wee1 Has an Essential Role in the Nuclear Divisions of Early Embryogenesis

Corresponding author: Shelagh D. Campbell, CW405, Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada., shelagh.campbell{at}ualberta.ca (E-mail)

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Drosophila, the maternally expressed mei-41 and grp genes are required for successful execution of the nuclear division cycles of early embryogenesis. In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1. We have identified mutations in a gene encoding a Cdk1 inhibitory kinase, Drosophila wee1 (Dwee1). Like mei-41 and grp, Dwee1 is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of Dwee1 mutants, as well as genetic interactions between Dwee1, grp, and mei-41 mutations, suggest that Dwee1 is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Cdk1 by Dwee1 is required for proper regulation of the early syncytial cycles of embryogenesis.

THE nuclear division cycles of early Drosophila embryogenesis provide a formidable challenge to the cell cycle regulatory machinery. Initially, these cycles consist of extraordinarily rapid oscillations between S phase (2–3 min) and mitosis (5–6 min) that utilize maternally provided protein and RNA and occur within a syncytium. Interphase length then increases progressively during cycles 10–13 prior to the midblastula (MBT) or maternal-zygotic (MZT) transition in cycle 14 that precedes the onset of gastrulation (FOE and ALBERTS 1983 ). During normal embryogenesis, the developmentally regulated lengthening of interphase during cycles 10–13 requires maternally provided mei-41 and grp encoded kinases (SIBON et al. 1997 , SIBON et al. 1999 ). These genes encode homologs of the evolutionarily conserved checkpoint kinases Rad3/ATM and Chk1 (HARI et al. 1995 ; FOGARTY et al. 1997 ). In fission yeast and humans, these kinases are components of a premitotic checkpoint that becomes activated in response to incompletely replicated or damaged DNA (JIMENEZ et al. 1992 ; WALWORTH et al. 1993 ; BEAMISH et al. 1996 ; BENTLEY et al. 1996 ; WALWORTH and BERNARDS 1996 ; SANCHEZ et al. 1997 ). Activation of the checkpoint is associated with inhibitory phosphorylation of Cdk1 (the central mitotic regulatory kinase) on a conserved tyrosine residue (Y15) by Wee1 kinases (O'CONNELL et al. 1997 ; RHIND et al. 1997 ; RHIND and RUSSELL 1998 ). In response to DNA damage, Chk1 becomes activated (in a Rad3-dependent fashion) and phosphorylates Cdc25 on a residue that promotes Cdc25 interaction with members of the 14-3-3 family of proteins (PENG et al. 1997 ; SANCHEZ et al. 1997 ; ZENG et al. 1998 ; CHEN et al. 1999 ; LOPEZ-GIRONA et al. 1999 ). This interaction prevents Cdc25 phosphatase from interacting with and activating Cdk1 by removal of Wee-catalyzed inhibitory phosphorylation. Consequently, either loss of Chk1 function or loss of Wee1 function can compromise the premitotic checkpoint.

The involvement of mei-41 and grp in the slowing of the early embryonic cycles is proposed to reflect activation of a DNA replication checkpoint once maternally provided replication functions become limiting (FOGARTY et al. 1997 ; SIBON et al. 1997 , SIBON et al. 1999 ). The conservation of these checkpoint genes among eukaryotes implies that the biochemical mechanism by which they function might also be conserved. If so, it suggests that Drosophila embryos employ inhibitory phosphorylation of Cdk1 by a Wee1-like kinase to coordinate this developmentally regulated checkpoint. Inhibitory phosphorylation of maternal Cdk1 is not detected in Drosophila embryo extracts during cycles 10–13 when the mei-41/grp pathway is required, however (EDGAR et al. 1994 ). This observation may indicate that Drosophila embryos employ the mei-41/grp pathway in a novel mechanism that is independent of Wee1-mediated inhibitory phosphorylation of Cdk1 to lengthen the syncytial cycles. We undertook a genetic analysis of Dwee1, a gene that encodes a Drosophila Cdk1 inhibitory kinase, to investigate these possibilities (CAMPBELL et al. 1995 ).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutagenesis screen for Dwee1 mutants:
cn/cn males were mutagenized with either ethyl methanesulfonate (EMS; 25–50 mM) or diepoxybutane (DEB; 5 mM) according to standard protocols and then mated en masse to Sco/CyO, cn virgin females. These flies were transferred onto fresh media daily for 4–5 days, whereupon mutagenized males were removed. F₁ progeny males carrying isolated mutagenized second chromosomes were collected and crossed individually to Df(2L)Dwee1^W05/CyO, cn virgin females (the origin and characterization of this deletion is described in the RESULTS). The F₂ progeny were then scored for presence of the cn/Df(2L)Dwee1^W05 class. Absence of this class indicated recovery of a zygotic lethal mutation (lethal alleles were designated DL or EL, depending on whether DEB or EMS was the relevant mutagen). In crosses where viable hemizygous F₂ progeny were obtained, females of this class were mated to siblings to test for fertility (female-sterile alleles were designated DS or ES, depending on whether DEB or EMS was the relevant mutagen). Mutant stocks were established by mating of retained cn/CyO, cn siblings. Identified mutants were then further classified by complementation crosses with known mutants in the region and with Df(2L)spd-J2, a deletion whose published breakpoints are 27C1-28A (NEUMANN and COHEN 1996 ). Our genetic and molecular analysis of a stock carrying this deletion suggests that the distal breakpoint of this aberration is actually 27C4-5.

Transgene rescue experiments:
A heat-shock-inducible construct was made by cloning the originally described Dwee1 cDNA into the pCaSpeR-hs vector (CAMPBELL et al. 1995 ). A genomic DNA construct that includes all of the Dwee1 coding region plus upstream and downstream flanking DNA was constructed by cloning a ~10-kb HindIII fragment (sequence coordinates 14,273–24,263 in the DS01321 clone shown in Fig 1) into a pUAS vector (BRAND and PERRIMON 1993 ). For the inducible Dwee1 rescue experiments, embryos were initially collected from mated Dwee1^ES1 hemizygous females carrying a hsDwee1 transgene for 4 days without heat shocks. No viable embryos were observed. Flies were then heat-shocked in a 37° water bath once daily as indicated, collecting embryos at ~24-hr intervals (specific conditions are available on request). Fixed embryos (37% formaldehyde:heptane for 3 min) were then stained with Hoechst 33258 for analysis. Expression of the transgene in Dwee1^ES1/hsDwee1, Df(2L)Dwee1^W05 adult females was confirmed by heat-shocking for 30 min (37°), with a 90-min recovery period. These flies were then homogenized in loading buffer (2.0% SDS, 60 mM Tris (pH 6.8), 0.01% bromophenol blue, 10% glycerol, and 0.1 M DTT) and the cleared extract was diluted in loading buffer prior to SDS-PAGE (8% acrylamide). Proteins were then transferred onto Hybond-P membrane and the membrane was probed with a rabbit anti-Dwee1 primary antibody (anti-DKD at 1:1600). The membrane was then reprobed with mouse anti-ß-tubulin (1:500; Amersham, Buckinghamshire, UK). Secondary antibody hybridization signal was detected using the ECL+ chemiluminescence system (Amersham).

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Genome organization of cytological region 27C4-5 with respect to transcribed genes and the extent and position of deletions and P-transposon insertions described in the text. The open horizontal boxes indicate the extent of the two deletions that impinge on this region, with shaded boxes representing regions within which the deletion endpoints were molecularly mapped by genomic hybridization analysis. The numbered coordinates represent genomic DNA sequence positions within the DS01321 clone. The positions of sites for restriction enzyme EcoRI are indicated (R) on the horizontal line representing the genomic sequence as a further orientation guide. Triangles connected to the sequence by vertical lines represent the insertion sites of two P-transposon insertion alleles of the hrp48 locus that are described in further detail in the text. BDGP determined the indicated cytological and molecular positions of these P-transposon insertions. Solid horizontal lines represent the exon/intron organization and orientation of transcripts identified in the region that correspond to previously sequenced ESTs in the BDGP database. ESTs with open reading frames whose translation products show high sequence similarity to proteins identified in other organisms are named with respect to those homologs. GH23590 and LD39226 represent ESTs that do not show significant homology to other known genes and may correspond to exons of the same gene. The transcript designated dhp1 is homologous to a gene implicated in recombination and RNA metabolism called dhp1/RAT1/Dhm1 in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and mice, respectively (KENNA et al. 1993 ; SHOBUIKE et al. 1995 ; SUGANO et al. 1994 ). The transcript designated NOP5 is homologous to a gene of the same name implicated in nucleolar assembly in S. cerevisiae (WU et al. 1998 ). The transcript designated RBP1-like is similar to a member of the SR protein family of splicing accessory factors required for sex-specific splicing of doublesex (HEINRICHS and BAKER 1995 ; LYNCH and MANIATIS 1996 ). The genes designated Dwee1 and hrp48 are described in further detail in the text.

Responses of Dwee1 mutants to hydroxyurea:
A genetic cross between Dwee1^ES1/CyO and Df(2L)Dwee1^W05/CyO flies was done, following which 24-hr embryo collections were made. After a further 24 hr of development, 1 ml of aqueous hydroxyurea concentrate was added to the media (values in Fig 5 indicate final concentration). Distilled water (1 ml) was substituted for controls. Adult flies were scored daily once they began to eclose, to completion.

View larger version (113K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Comparison of nuclei from equivalently staged wild-type and Dwee1 mutant embryos. (A) Nuclei from a wild-type embryo in interphase of cycle 13. (B) Nuclei from a Dwee1-derived embryo in interphase of cycle 13. The arrowheads indicate nuclei that have failed to divide in mitosis 12. Bar, 12 µm.

View larger version (108K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Expression of a hsDwee1 transgene can rescue the maternal lethal phenotype of Dwee1 mutant-derived embryos. (A) Immunoblot showing induction of Dwee1 protein by heat shock of Dwee1ES1/hsDwee1, Df(2L)Dwee1W05 adult female flies. (B) Classes of embryos produced by Dwee1ES1/hsDwee1, Df(2L)Dwee1W05 mothers. Numbers of embryos counted for each collection are given at the bottom of the graph. The bar marked "control" represents embryos collected prior to induction of the transgene. Embryos that were completely syncytial and displayed the typical nuclear defects described for Dwee1 mutants were scored as "arrested." Embryos were scored as rescued if they were able to complete syncytial development and cellularize (at least partially). Embryos that were developmentally too early to score as being either arrested or rescued were scored as "pre-arrest." (C) Variation in embryonic phenotypes observed in the heat-shock rescue experiment. (i) Syncytial arrest phenotype typical of Dwee1-derived embryos. (ii) Mosaic embryo with both cellularized (right) and arrested (left) domains. (iii and iv) Apparently normal late embryo and early larva, respectively. Bar, 62 µm.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Location of molecular lesions identified in genomic DNA of the Dwee1 mutants identified in this study. The sequence shown represents amino acid residues 246 to 486 of the Dwee1 coding region and the letters denote amino acids in the standard single-letter designation. Letters in bold signify residues that are conserved in other Wee1 kinases. The arrows indicate the position of lesions identified in the Dwee1ES1 (ES1) and Dwee1ES2 (ES2) EMS-induced and Dwee1DS1 (DS1) DEB-induced mutant alleles, respectively, and point to the altered residues that the mutant alleles are predicted to encode.

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Dwee1 mutants are defective for a zygotic DNA replication checkpoint. The graph shows populations of progeny from Dwee1ES1/CyO flies crossed with Df(2L)Dwee1W05/CyO flies that were grown in vials containing the indicated concentration of hydroxyurea. The visible adult marker Cy carried on the second chromosome balancer in the heterozygotes was used to distinguish the genotypes of hemizygous and heterozygous flies.

Genomic sequencing and DNA analysis:
Genomic DNA was extracted by standard techniques (CAMPBELL et al. 1995 ) from hemizygous adult flies and used as a template for direct amplification using Pfu polymerase. The amplified product was then cycle-sequenced on both strands using the Thermosequenase system (Amersham). Mutations were confirmed by independent sequencing reactions. The GenBank accession number for Dwee1 is U17223. Genomic DNA analysis to localize insertions and deletion breakpoints was also done as described in CAMPBELL et al. 1995 , using digoxygenin-labeled probes according to the manufacturer's recommendations (Boehringer Mannheim, Indianapolis).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A screen for lethal and sterile mutations in genes uncovered by Df(2L)Dwee1^WO5:
Previous studies of the Dwee1 locus utilized a large deletion, called Df(2L)Dwee1;27A-28B, that uncovers Dwee1 as well as a number of other genes (CAMPBELL et al. 1995 ). These studies established that loss of zygotic Dwee1 function does not produce a detectable mitotic phenotype in embryos. To generate a smaller deletion for further genetic studies of the locus, transposase-mediated imprecise excision of a nearby P{w+} transposon insertion, associated with l(2)k10413, was used to generate w- derivative chromosomes (see Fig 1). Both l(2)k10413 and a nearby P insertion, l(2)02647, have been cytologically mapped to position 27C4-5 by the Berkeley Drosophila Genome Project (BDGP) and have been described as mutant alleles of the hrp48 gene (also called Hrb27C) that encodes a ribonuclear splicing factor (MATUNIS et al. 1992 ; HAMMOND et al. 1997 ). Flies that are transheterozygous for these two alleles (l(2)k10413/l(2)02647) are viable but usually have small nicks in the wing margin. The w- derivatives generated by mobilization of the P(w+) insertion associated with l(2)k10413 were thus initially screened by failure to complement l(2)02647 for viability as a means of identifying potential excision events that extended toward the Dwee1 locus. Stocks carrying noncomplementing alleles were then screened by genomic DNA analysis for molecular aberrations that would indicate the recovery of deletions uncovering the Dwee1 coding region. A single deletion, Df(2L)Dwee1^WO5, was identified by this approach. This deletion uncovers the Dwee1 locus and additional genes shown in Fig 1 that were identified by sequence comparisons of the completed genomic sequence of the region (P1 clone: DS01321, Berkeley Drosophila Genome Project) with the expressed sequence tag (EST) database. Df(2L) Dwee1^WO5 is lethal in transheterozygous combinations with all previously characterized alleles of hrp48 and with the single P-insertion allele of another gene of unknown function designated l(2)k00213 (mapped to position 27C2-3 by BDGP). It also fails to complement Df(2L)spd-J2 (NEUMANN and COHEN 1996 ). Homozygous Df(2L)Dwee1^WO5 mutants derived from heterozygous parents die late in embryogenesis with no obvious mitotic abnormalities, consistent with previous characterization done with the larger deletion, Df(2L)Dwee1;27A-28B. A recombinant chromosome carrying Df(2L)Dwee1^WO5 and a proximal flipase recombinase target (FRT) sequence was generated to investigate maternal requirements for Dwee1 function (CHOU and PERRIMON 1996 ). No eggs were generated from FLP-expressing, ovo^D FRT/Df(2L)Dwee1^WO5FRT transheterozygous females for analysis, however, from which we could only conclude that one of the genes uncovered by the deletion must be essential for oogenesis (data not shown).

To further investigate the function of Dwee1, a chemical mutagenesis screen was devised to identify point mutations in genes within the region delimited by Df(2L)Dwee1^WO5. A standard F₂ screening protocol was followed as described in MATERIALS AND METHODS. Hemizygous F₂ progeny were tested for zygotic lethality and those that were viable were then tested for female sterility. By this approach we identified four lethal and three female-sterile mutants from progeny representing ~4500 individual candidate chromosomes.

Characterization of lethal alleles recovered in the screen:
Complementation tests established that the first lethal mutation recovered in our screen (l(2)EL1) is an allele of the gene thought to be associated with the P-transposon insertion designated l(2)k00213 (TOROK et al. 1993 ). We have not yet determined which gene within or overlapping Df(2L)Dwee^WO5 is affected by these mutations, but there are clearly several potential candidates (Fig 1).

Df(2L)spd-J2, previously characterized as uncovering cytological interval 27C1-28A, was crossed to all of the mutations recovered in our screen as well as to known P-element mutations in the region (NEUMANN and COHEN 1996 ). This deletion failed to complement lethal P-insertion alleles of the hrp48 locus [l(2)02647 and l(2)k10413] as well as our new lethal mutation called l(2)EL2, but does complement all other mutations described in this study except Df(2L)Dwee1^WO5. These observations prompted us to determine the distal breakpoint of this deletion using molecular techniques. We found it within an interval just distal to hrp48, as indicated in Fig 1. The complementation patterns of two other lethal alleles recovered in our screen, l(2)EL3 and l(2)DL1, are complex (see Table 1). Both complement the two P-insertion alleles of hrp48 as well as Df(2L)spd-J2, suggesting that these mutations have not disrupted the hrp48 locus. However, our new hrp48 allele l(2)EL2 failed to complement l(2)EL3. Furthermore, while l(2)DL1 complemented all the hrp48 alleles we tested, it failed to complement l(2)EL3. We are not able to resolve the genetic basis for these peculiar genetic interactions at this time. One possibility is that our data reflect interallelic complementation among different alleles of a complex genetic locus. Various cDNA clones characterized by the BDGP from the hrp48 locus fall into three distinct classes of splice products, possibly accounting for the observed genetic complexity (see Fig 1).

Characterization of Dwee1 female-sterile mutations recovered in the screen:
Complementation tests showed that all three female-sterile mutations recovered in our screen (Dwee1^ES1, Dwee1^ES2, and Dwee1^DS1) are alleles of the same gene, and data described later in this section establish that this gene corresponds to Dwee1. We undertook a detailed phenotypic analysis of one of the mutant alleles, Dwee1^ES1. Hemizygous Dwee1^ES1 mutant females are viable but completely sterile and show no paternal rescue effect (hemizygous males are fertile, however). Hemizygous females lay abundant eggs of normal appearance that proceed through the early syncytial nuclear cycles without incident. During cycles 11 and 12, however, nuclei in mutant-derived embryos fail to separate at the end of mitosis and remain fused (Fig 2). This phenotype and the subsequent clumping and fragmentation of nuclei that we observe (Fig 3C) is identical to what is seen in embryos collected from grp or mei-41 mutant females (FOGARTY et al. 1994 ; SIBON et al. 1999 ). This observation suggests a possible role for Dwee1 in the same developmental checkpoint as mei-41 and grp.

We undertook two different approaches to demonstrate that the complementation group represented by the three female-sterile mutations does in fact correspond to Dwee1. First, we sequenced genomic DNA isolated from adults hemizygous for each of the alleles (Dwee1^ES1, Dwee1^ES2, and Dwee1^DS1), covering the entire transcribed region and ~150 bp of flanking DNA at each end. For comparison, we sequenced genomic DNA from the stock used to generate the mutants. With respect to their maternal phenotype, Dwee1^ES1 and Dwee1^ES2 behave as classical amorphic alleles (MULLER 1932 ). Genomic DNA isolated from each of these mutants contains a DNA lesion within the kinase domain of Dwee1 that is expected to either abolish or severely disrupt the function of the gene (Fig 4). Dwee1^ES1 contains an 8-bp deletion causing a frameshift followed by a stop codon, truncating the protein in kinase domain IV. Dwee1^ES2 contains a missense mutation that changes a glutamate residue that is conserved among Wee1-like kinases to a lysine at position 308 in the protein (E308K). Dwee1^DS1 behaves as a classical hypomorphic allele in that the phenotype of embryos derived from homozygous females is much less severe (many cellularize and some even develop to adulthood) than that of embryos derived from hemizygous females (which rarely cellularize and never hatch). Sequence analysis of this allele showed that it contains a missense mutation changing a conserved phenylalanine residue to isoleucine at amino acid residue 250 within the ATP-binding site of the protein (F250I). Presumably this lesion is still compatible with low-level function of the protein. The Dwee1^ES1 allele shows an antimorphic interaction with the Dwee1^DS1 allele in that the phenotype of embryos derived from Dwee1^DS1/Dwee1^ES1 transheterozygous mothers is more severe (embryos never cellularize) than seen in Dwee1^DS1/Df(2L)Dwee1^WO5 hemizygotes. Conceivably, this reflects titration of positive regulatory factors by the truncated Dwee1^ES1 protein, thus lowering the effective levels of Dwee1^DS1 function.

We were also able to partially rescue the phenotype of mutant embryos with a heat-inducible Dwee1 cDNA transgene. Maternal Dwee1^ES1 hemizygous flies carrying this transgene were briefly heat-shocked to induce expression as confirmed by immunoblot analysis (Fig 3A). Rescue was scored as development at least to the cellularization stage (cycle 14), which mutant-derived embryos otherwise never reach. By this measure, ~50% of the embryos could be rescued by maternal expression of the transgene. Cessation of heatshocks produced a decline in numbers of rescued embryos (Fig 3B). We observed wide phenotypic variation in the extent of phenotypic rescue, presumably reflecting variations in the amount and timing of Dwee1 protein and mRNA deposited into individual eggs. These ranged from mosaic embryos containing both cellularized and syncytial sectors to apparently normal late embryos and first instar larvae that were nonetheless unable to complete development (Fig 3C). In contrast, heat-shock treatment of Dwee1^ES1 hemizygous females lacking the transgene produced no cellularized embryos, eliminating the possibility that our mutants were being rescued by the experimental protocol alone. Furthermore, a single transgene copy of a genomic DNA construct that contains Dwee1 coding sequences plus flanking DNA (and includes the adjacent dhp1-like gene) can completely rescue the maternal lethal phenotype. These two lines of evidence demonstrate that molecular lesions consistent with loss of function in Dwee1 are found in the female-sterile mutants and also show that Dwee1 expression is both necessary and sufficient to rescue the maternal lethal phenotype. We conclude from this evidence that we have identified mutant alleles of Dwee1. The striking similarity between the phenotype of Dwee1 mutant-derived embryos and embryos derived from grp or mei-41 mutants provides a strong argument that maternally provided Dwee1 plays an essential role in the same developmental process as grp and mei-41.

Additional evidence in favor of this hypothesis is afforded by providing extra maternal copies of the genomic Dwee1 transgene in a mei-41^D3 mutant background. Females homozygous for the mei-41^D3 allele produce cellularized embyros at a very low frequency (2%, N = 106). The frequency of cellularized embryos is dramatically increased by adding an extra maternal copy of a Dwee1 genomic transgene (20%, N = 109). The mei-41^D3 mutant embryos are further rescued by addition of two Dwee1 transgenes (50%, N = 72), to the extent that some mei-41^D3-derived embryos were able to develop to adulthood. In contrast, parallel experiments in a grp¹ background did not produce any rescue of the mutant phenotype with either one or two extra copies of Dwee1. The simplest interpretation we can offer for why the results differ between grp and mei-41 mutants in these experiments is that the mei-41^D3 is not a complete loss-of-function allele, and consequently mei-41^D3 mutants are more sensitive to increased dosage of Dwee1 than grp¹ mutants. Alternatively, grp may respond to two different signaling pathways whereas mei-41 may respond to only one of the two. Dwee1 overproduction could be sufficient to rescue the common function but not the grp-specific one according to this model. Another test for functional interactions among these genes was to assess the effect of lowering the maternal dosage of mei-41+ or grp+ in a homozygous Dwee1^DS1 maternal background. The incompletely penetrant syncytial arrest phenotype of homozygous Dwee1^DS1-derived embryos (54% cellularized, N = 107) was enhanced by subtracting a maternal copy of mei-41+ (39%, N = 141). Removal of one maternal copy of grp+ produced an even greater enhancement of the mutant phenotype of Dwee1^DS1 embryos (29% cellularized, N = 127).

We wanted to assess whether Dwee1 hemizygous flies derived from heterozygous parents were capable of mounting an effective response to delays in DNA replication, since the slowing of the late syncytial cycles has been proposed to reflect activation of a DNA replication checkpoint (SIBON et al. 1999 ). For this experiment, we assessed the sensitivity of Dwee1^ES1 hemizygous larvae to treatment with hydroxyurea (HU), a drug that inhibits DNA replication. In fission yeast, the "checkpoint rad" group of mutants as well as wee1 mutants are all extremely sensitive to HU. In Drosophila, mei-41 and grp mutant larvae also exhibit this response (SIBON et al. 1999 ). Genetic crosses between balanced heterozygous stocks carrying either the Dwee1^ES1 mutant chromosome or the Df(2L)Dwee1^W05 chromosome generate both heterozygous and hemizygous viable adult progeny. Exposure to 1 or 2 mM HU eliminated the hemizygous Dwee1^ES1 class of progeny, indicating that Dwee1 mutant larvae are indeed highly sensitive to HU, presumably reflecting a requirement for Dwee1 activity in a fully functional DNA replication checkpoint (Fig 5).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

These studies establish that Dwee1 has an essential maternal function during the nuclear division cycles of embryogenesis and also implicate zygotic Dwee1 function in a cell cycle checkpoint that responds to inhibition of DNA replication. The demonstration that Dwee1 has a role during the early syncytial nuclear cycles calls into question a previous assumption that inhibitory phosphorylation does not control these cycles. Analyses of the state of phosphorylation during the early cycles had failed to detect inhibitory phosphorylation of Cdk1 prior to cycle 13 (EDGAR et al. 1994 ). Furthermore, because reduction in the gene dose of cyclin A and cyclin B slowed the late nuclear cycles, it was suggested that progress of these cycles is regulated by accumulation of cyclins to a threshold level. The finding that Dwee1 is required for completing the nuclear division cycles suggests that inhibitory phosphorylation does play a role in their regulation after all. The failure to detect inhibitory phosphorylation during these cycles can be explained if only a small pool of Cdk1 is subject to this modification. Wee1-type kinases are predominantly nuclear in Drosophila and other organisms and nuclear Wee1 activity is sufficient to block entry into mitosis even in the presence of high cytoplasmic Cdk1 activity (HEALD et al. 1993 ; CAMPBELL et al. 1995 ). Hence, we suggest that inhibitory phosphorylation of a small nuclear pool of Cdk1 contributes importantly to the control of the syncytial cycles. The proposal that inhibitory phosphorylation regulates syncytial cycles was an implicit component of a recently proposed model for the mechanism by which mei-41 and grp regulate the progressive lengthening of these cycles (SIBON et al. 1997 , SIBON et al. 1999 ). In response to incompletely replicated DNA, the recognized activities of these conserved checkpoint kinases arrest the cell cycle by preventing the removal of inhibitory phosphates from Cdk1. While this model appears to be at odds with the lack of detectable inhibitory phosphorylation of Cdk1 during the syncytial cycles (EDGAR et al. 1994 ), our findings that Dwee1 is required for the early nuclear division cycles supports this proposal. Indeed, the apparent parallels in the phenotypes of mei-41, grp, and Dwee1 maternal mutants suggest that these genes operate by a similar mechanism. Because our results implicate this pathway without defining precisely how it is induced, it remains possible that the same pathway could be used in a unique regulatory circuit, as was recently suggested (SU et al. 1998 ). In either case, the lesson seems to be that the remarkable conservation of the eukaryotic cell cycle regulatory machinery is coupled with an equally remarkable flexibility in how that machinery can be deployed, depending on the particular developmental constraints of each organism. In early Drosophila embryos, a regulatory pathway that usually serves a surveillance function plays an essential cell cycle role.

It was unexpected that zygotic Dwee1 function would be dispensable under normal growth conditions, since Cdk1 inhibitory phosphorylation appears to play an important role in cell cycle regulation at many stages of development in Drosophila. Following the last syncytial division during interphase of cycle 14, Cdk1 becomes quantitatively inhibited by phosphorylation (EDGAR et al. 1994 ). This dramatic regulatory transition could result from delocalization of Dwee1, activation of a cytoplasmically localized Cdk1 inhibitory kinase, inhibition of cytoplasmic Cdc25, or more active exchange of Cdk1 between the nucleus and cytoplasm during cycle 14. We are currently investigating these possibilities. It has been demonstrated that entry into mitosis 14 depends on zygotic expression of Cdc25^stg phosphatase and removal of inhibitory phosphate from Cdk1 (EDGAR and O'FARRELL 1989 , EDGAR and O'FARRELL 1990 ). Furthermore, Cdc25^stg activity is also required during the following postblastoderm mitoses of embryogenesis and during imaginal disc development (EDGAR and O'FARRELL 1989 , EDGAR and O'FARRELL 1990 ; MILAN et al. 1996 ; JOHNSTON and EDGAR 1998 ). Cdc25^twe activity is also required during meiosis (ALPHEY et al. 1992 ; COURTOT et al. 1992 ). These requirements for Cdc25^stg imply that inhibitory phosphorylation is normally significant at all of these stages of development. In fission yeast, loss of Wee1 kinase can suppress requirements for the Cdc25 phosphatase. In Drosophila, however, loss of zygotic Dwee1 function does not bypass the requirement for Cdc25^stg activity (CAMPBELL et al. 1995 ). The continued requirement for Cdc25^stg activity might be due to maternal perdurance of Dwee1 function. Alternatively, there might be other Wee1 kinases that can function either redundantly with Dwee1 or independently. We have recently cloned the gene encoding a Drosophila homolog of Myt1, a Wee1-related kinase, which may contribute to some of these activities (S. CAMPBELL, unpublished data; MUELLER et al. 1995 ).

	ACKNOWLEDGMENTS

Assistance by Ellen Homola in making transgenic flies is gratefully acknowledged. Christine Walker sequenced the Dwee1^DS1 allele. The Berkeley Drosophila Genome Project (Todd Laverty and Amy Beaton in particular) and the Bloomington Stock Center provided fly stocks and genomic information used in this work. Thanks also to BDGP for the sequence of the genomic clone DS01321. This work was funded by research grants from the Alberta Heritage Foundation for Medical Research (AHFMR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Medical Research Council of Canada (S.D.C), by the National Institutes of Health (P.O'F.), and by graduate student fellowships from AHFMR and NSERC (D.P.).

Manuscript received November 29, 1999; Accepted for publication January 13, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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