Ectopic Expression of the Drosophila Cdk1 Inhibitory Kinases, Wee1 and Myt1, Interferes With the Second Mitotic Wave and Disrupts Pattern Formation During Eye Development

Corresponding author: Shelagh D. Campbell, University of Alberta, CW405, Edmonton, AB 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

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis.

THE control of mitosis by inhibitory phosphorylation of cyclin-dependent kinase (Cdk)1 has been characterized extensively in unicellular eukaryotes. In Schizosaccharomyces pombe, signaling pathways responsive to cell size, DNA damage, and DNA replication target the phosphorylation of Cdk1 residue tyrosine 15 (Y15), thereby functioning to maintaining genome integrity (RHIND et al. 1997 ; RHIND and RUSSELL 1998 ). Inhibitory phosphorylation of Cdk1 is catalyzed by both Wee1 and Mik1 kinases in S. pombe (RUSSELL and NURSE 1987B ; FEATHERSTONE and RUSSELL 1991 ; LUNDGREN et al. 1991 ; LEE et al. 1994 ) and is reversed by Cdc25 and Pyp3 phosphatases (RUSSELL and NURSE 1986 ; GOULD et al. 1990 ; MILLAR et al. 1991 , MILLAR et al. 1992 ). In contrast, inhibitory phosphorylation of a Cdk1 homolog (CDC28) is not required for maintenance of genome integrity in Saccharomyces cerevisiae (AMON et al. 1992 ; SORGER and MURRAY 1992 ). Instead, a SWE1-mediated checkpoint delays mitosis by inhibiting CDC28 in response to defective assembly of the actin cytoskeleton and promotes filamentous growth when nutrients are limiting (LEW and REED 1995 ; SIA et al. 1996 , SIA et al. 1998 ; MCMILLAN et al. 1998 ; BARRAL et al. 1999 ; EDGINGTON et al. 1999 ).

During Drosophila embryogenesis, inhibitory phosphorylation of Cdk1 is required for maintaining G2 phase during the embryonic cell divisions. Expression of cdc25^string overcomes this inhibition, inducing mitosis in spatially and temporally patterned mitotic domains (EDGAR and O'FARRELL 1990 ). The intricate pattern of cdc25^string transcription is governed by cis elements in a large regulatory region that integrates a diverse array of patterning gene inputs to direct the appropriate spatiotemporal pattern of cdc25^string expression during embryonic and imaginal development (EDGAR et al. 1994 ; JOHNSTON and EDGAR 1998 ; LEHMAN et al. 1999 ). Heat shock expression of a constitutively active, nonphosphorylatable Cdk1 variant (Cdk1AF) is lethal to Drosophila embryos, indicating that inhibitory phosphorylation of Cdk1 is essential for regulating mitosis during development; however, regulation of a similar S phase kinase (Cdk2) on a conserved tyrosine residue is not (LANE et al. 2000 ).

In metazoans, two adjacent inhibitory phosphorylation sites on Cdk1 (T14 and Y15) are substrates for two distinct Wee1-like kinases that differ in their subcellular localization. Nuclear Wee1 kinases phosphorylate Y15 exclusively, whereas Myt1, a membrane-localized Wee1-like kinase, can phosphorylate either site (KORNBLUTH et al. 1994 ; MUELLER et al. 1995 ; BOOHER et al. 1997 ; LIU et al. 1997 ). The physiological significance of these differences between the Wee1 and Myt1 kinases is presently unknown. We are addressing this question by characterizing the functions of Wee1 and Myt1 kinases during Drosophila development. Drosophila encodes a single wee1 homolog (Dwee1), originally identified by its ability to complement a lethal mitotic catastrophe phenotype in S. pombe cells that were mutant for both wee1 and mik1 (CAMPBELL et al. 1995 ). Null alleles of Dwee1 are maternal effect lethal and Dwee1-derived embryos undergo catastrophic nuclear defects during the late syncytial divisions that include failure to complete nuclear division (PRICE et al. 2000 ) and failure to lengthen interphase, as normally occur when a developing embryo approaches cycle 14 (D. PRICE, unpublished data). The phenotype of Dwee1-derived mutant embryos is similar to phenotypes of maternal mutants for mei-41 or grapes (grp), the Drosophila homologs of the checkpoint kinases rad3/ATR and chk1, respectively (FOGARTY et al. 1994 , FOGARTY et al. 1997 ; SIBON et al. 1997 , SIBON et al. 1999 ). These phenotypic similarities suggest that the three genes act in a common checkpoint pathway during early embryonic development, an idea supported by genetic interactions between mutant alleles of these genes (PRICE et al. 2000 ).

Given the critical importance of inhibitory phosphorylation during embryogenesis, it was puzzling that the zygotic function of Dwee1 is not essential and that Dwee1 mutants develop normally under ordinary circumstances. Dwee1 mutant larvae do die when they are fed hydroxyurea at concentrations that wild-type larvae can tolerate, however, apparently due to a defective DNA replication checkpoint (PRICE et al. 2000 ). The viability of zygotic Dwee1 mutants could be due to the presence of a redundant Cdk1 inhibitory kinase such as Myt1. Although cellular localization and substrate specificity differences suggest that Wee1 and Myt1 homologs serve distinct roles in cell cycle regulation, the two metazoan Wee1-like kinases may also share some redundant functions, as wee1 and mik1 do in S. pombe (LUNDGREN et al. 1991 ). To investigate this possibility we cloned the single Myt1-like gene from Drosophila, Dmyt1, and are undertaking a genetic analysis of its function during development.

In this report we describe phenotypic defects caused by overexpressing either Dwee1 or Dmyt1 in developing tissues. Overexpression in the eye imaginal disc causes visible defects in the adult eye. The eye phenotype can be modified by mutations in known cell cycle regulators, suggesting that this system might be capable of detecting previously uncharacterized mitotic regulators that have evolved to coordinate cell proliferation with specific developmental events. We have tested this idea by screening for dominant genetic modifiers, using a collection of deletions comprising 70–80% of the Drosophila euchromatic genome. These tests have identified several loci that potentially encode novel regulators of either Wee1 or Myt1.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cloning of the Drosophila Myt1 gene:
A small fragment of Dmyt1 was amplified by PCR using degenerate primers designed against conserved regions of Xenopus and human Myt1 (CKLGDFG and AADVFSL). After sequencing to confirm that we had in fact isolated a genomic sequence that was similar to the Myt1 homologs, the PCR fragment was labeled and used to screen the pNB embryonic cDNA library (BROWN and KAFATOS 1988 ). We were unsuccessful in isolating a cDNA clone by this approach, so we designed a reverse primer specific to the cloned Dmyt1 fragment and used it in combination with a pNB vector primer to PCR amplify the 5' end of a cDNA sequence from the same library. The fragment obtained was cloned and sequenced and the information was used to identify two cDNA clones from the Berkeley Drosophila EST Project database (GH08848 and LD34963). These clones were both fully sequenced and found to include identical coding regions that show significant sequence similarities to human and Xenopus Myt1 within the predicted kinase domain (LD34963 is 20 bp longer at the 5' end, but the sequences are otherwise identical except for the length of the poly(A) tail at the 3' end). The complete molecular characterization of the Dmyt1 gene will be presented elsewhere (Z. JIN, S. RABINOVITCH and S. D. CAMPBELL, unpublished results).

Generation of Dwee1 and Dmyt1 transgenic stocks:

• pUAST-Dwee1 and pUAST-Dmyt1: To synthesize pUAST-Dwee1, a 2.2-kb Dwee1 cDNA fragment was excised from pBluescript SK(+) by KpnI/NotI digestion and subcloned into the pUAST vector using the same restriction sites (BRAND and PERRIMON 1993 ). pUAST-Dmyt1 was constructed by cloning a 1.9-kb EcoRI/XhoI fragment that includes the entire Dmyt1 cDNA from LD34963 and inserting it into the pUAST plasmid vector, also cut with the same restriction enzymes.

• pUASp-Dwee1 and pUASp-Dmyt1: The 2.2-kb KpnI/NotI Dwee1 cDNA fragment (as above) was inserted into the pUASp vector (RORTH 1998 ) cut with the same restriction enzymes. A PCR-amplified Dmyt1 cDNA from the LD34963 clone containing KpnI/NotI linker restriction sites was cloned into the pUASp vector. This clone was then sequenced to establish that no new mutations were introduced during PCR amplification.

• pGMR-Dwee1 and pGMR-Dmyt1: The glass multimer reporter plasmid (pGMR; HAY et al. 1994 ) was cut with HpaI and NotI. The Dwee1 and Dmyt1 cDNAs were isolated from pUASp vector constructs by cutting with KpnI, blunting with T4 DNA polymerase, digestion with NotI, and then gel purification. Insert and vector were joined with T4 DNA ligase and the products verified by colony PCR. The transgene constructs were then injected into y w Drosophila embryos, using a 2-3-helper plasmid.

Scanning electron microscopy:
Flies of the desired genotypes were collected several days after eclosion, fixed, dehydrated, and critical-point dried essentially as described in SULLIVAN et al. 2000 . Critical-point-dried flies were then either imaged directly with a Philips (Cheshire, CT) ESEM (model XL30 ESEM ODP) or sputter-coated with gold and imaged with a Jeol (Tokyo) scanning electron microscope (SEM; model JSM-630FXV).

Transmission electron microscopy:
Fly heads of the desired genotypes were collected, fixed, and dehydrated as described in SULLIVAN et al. 2000 . Dehydrated heads were embedded in Spurr resin (SPURR 1969 ) with propylene oxide used as a transition solvent. Embedded heads were sectioned to 60 nm thickness with a Diatome diamond knife using a Reichert-Jung ultramicrotome (model ULTRACUT E). Sections were collected in water on copper grids, stained with uranyl acetate and lead citrate, and viewed on a Philips transmission electron microscope (TEM; model Morgagni 268). Images were collected with a Soft Imaging System digital camera (model Megaview II).

Immunochemistry:
Imaginal discs were fixed in 4% formaldehyde in PBS for 30 min at room temperature. Following fixation, the peripodial membrane was removed from the eye discs using tungsten needles. After blocking in 10% normal goat serum (NGS) made with PBS + 0.1% Tween-20 (PBT), the fixed discs were washed three times for 5 min in PBT and incubated at 4° overnight in primary antibody (rabbit antiphosphohistone H3; Upstate Biochemicals) at 1/600 dilution in 10% NGS. Discs were then washed four times for 10 min in 5% skim milk in PBT and incubated in preabsorbed secondary antibody (goat anti-rabbit conjugated with FITC; Jackson Immunoresearch, West Grove, PA) at 1/1000 dilution. Stained discs were washed four times for 10 min in PBT, stained with Hoechst 33258, and washed again in PBT. Eye discs were then separated from the optic lobe and mounted in 80% glycerol. Images were obtained on a Zeiss (Thornwood, NY) Axioskop 2 microscope equipped with a Photometrics (Tucson, AZ) SenSys digital camera.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Ectopic expression of Dwee1 in developing imaginal tissues:
To examine the consequences of overexpressing Dwee1 and Dmyt1 in different tissues, we generated transgenic lines that can express either gene under control of the Gal4/UAS system, as described in MATERIALS AND METHODS (BRAND and PERRIMON 1993 ). Fig 1 shows the effect of Gal4-induced expression of UAS-Dwee1 in various tissues (hereafter "UAS" refers to the UAST constructs). The pannier-Gal4 (pnr-Gal4) and apterous-Gal4 (ap-Gal4) drivers are each expressed in the developing dorsal thorax (CALLEJA et al. 1996 ). When either of these Gal4 drivers is combined with one copy of UAS-Dwee1, reduced numbers of sensory bristles are seen on the dorsal thorax, compared to wild type (Fig 1A, Fig B, and Fig D). Flies with ap-Gal4-driven UAS-Dwee1 also have upturned wings, suggesting that the dorsal compartment of the wing is smaller than the ventral compartment, consistent with these cells undergoing fewer cell divisions (data not shown). When two copies of the UAS-Dwee1 transgene are driven by either ap-Gal4 or pnr-Gal4, the bristle effects are more extreme and the dorsal epidermis is distorted, indicating that the phenotypic effects are sensitive to gene dosage (Fig 1C and Fig E). Combination of the ap-Gal4 driver with two copies of UAS-Dwee1 yields a nearly bald dorsal thorax accompanied by a severe reduction of the scutellum (Fig 1C). A more extreme phenotype is seen when the pnr-Gal4 driver is combined with two copies of UAS-Dwee1, producing a furrowed thorax, as if the two halves have failed to fuse properly (Fig 1E). This observation suggests that fusion may require temporally or spatially regulated cell divisions that can be blocked by our overexpression system. In the wing, UAS-Dwee1 combined with a wing-specific sd-Gal4 driver line produces extensive scalloping of the wing margin (Fig 1F) and an additional copy of UAS-Dwee1 (Fig 1G) also increases the severity of this mutant phenotype.

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Aberrant phenotypes caused by Dwee1 overexpression. (A) Thorax of a wild-type fly. (B) Thorax of a fly with a single copy of UAS-Dwee1 driven by a single copy of ap-Gal4. (C) Thorax of a fly with two copies of UAS-Dwee1 and a single copy of ap-Gal4. (D) Thorax of a fly with a single copy of UAS-Dwee1 and a single copy of pnr-Gal4. (E) Thorax of a fly with two copies of UAS-Dwee1 and a single copy of pnr-Gal4. (F) Wing of a fly with a single copy of UAS-Dwee1 and a single copy of sd-Gal4. (G) Wing of a fly with two copies of UAS-Dwee1 and a single copy of sd-Gal4.

Ectopic Dwee1 expression in the eye produces a rough eye phenotype (Fig 2). In Fig 2A and Fig B, are controls showing a wild-type eye and an eye from a fly with a single copy of the ninaE-Gal4 driver, respectively (FREEMAN 1996 ). When UAS-Dwee1 is combined with the ninaE-Gal4 driver, the eye facets are disorganized and frequent duplications of bristles are observed (Fig 2C). ninaE-Gal4 overexpression of Dmyt1 produced a similar phenotype (not shown). The Dwee1 and Dmyt1-induced rough eye phenotypes suggested to us that we could use Dwee1 or Dmyt1 transgenic flies in an assay system for identifying negative or positive regulators of mitosis, as described below.

View larger version (67K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Effects of Dwee1 overexpression on the adult eye as visualized by SEM. (A) A single copy of the ninaE-Gal4 driver transgene. (B) A single copy of the UAS-Dwee1 transgene. (C) A single copy of UAS-Dwee1 driven by a single copy of the ninaE-Gal4 transgene.

Genetic interactions with GMR-Dwee1 and GMR-Dmyt1:
The GMR overexpression vector uses a Glass transcription factor-binding enhancer to direct transgene expression posterior to the morphogenetic furrow (MF) in the developing eye (HAY et al. 1994 ). This single component system thus provides a convenient tool for rapidly testing genetic interactions. After cloning the cDNAs for each gene into this vector, we observed that GMR-Dwee1 and GMR-Dmyt1 transgenic lines each show dosage-sensitive rough eye phenotypes. In 12 independent transgene lines examined for each construct, the Dmyt1-induced phenotypes are consistently stronger than the Dwee1-induced phenotypes, suggesting a stronger effect of Myt1 on eye development that is not attributable to chromosomal position effects (data not shown). In Fig 3B we show an adult eye from a fly carrying four copies of GMR-Dmyt1, compared with a wild-type control eye (Fig 3A). Posterior to the MF, the second mitotic wave (SMW) generates a pool of uncommitted cells for recruitment into the developing ommatidial preclusters (WOLFF and READY 1991 ). To test our assumption that the aberrant phenotypes we observe when Wee1 or Myt1 are overexpressed are a consequence of inhibiting or delaying cell divisions required for normal development, we examined mitotic activity in eye imaginal discs isolated from a GMR-Dmyt1 transgenic strain. Fig 3C shows mitotic activity in a wild-type third larval instar eye disc, visualized by antibody staining for phosphohistone H3. In discs isolated from a GMR-Dmyt1 transgenic line, mitoses in the SMW are both reduced in number and delayed (inferred from the increased distance of mitotic cells from cells of the "first mitotic wave"; Fig 3D) when compared to wild type. Mitoses ahead of the morphogenetic furrow (the first mitotic wave) are unaffected by GMR-Dmyt1, as expected since GMR-driven expression does not occur in this region of the disc (HAY et al. 1994 ). We also observed that the ommatidial preclusters in the GMR-Dmyt1 flies appear disorganized when visualized by transmission electron microscopy of sectioned adult eyes. Fig 3E and Fig F, shows the effects of GMR-Dmyt1 on the arrangement of photoreceptor cells. Most of the identifiable cell types in the ommatidia appear to be present, although the arrangement and size of the rhabdomeres are often irregular. The GMR-Dmyt1 photoreceptor cell clusters often contain too few or too many cells, however, and there is a striking disruption of the regular hexagonal array of secondary and tertiary pigment cells that normally forms an interface between adjacent ommatidia (compare Fig 3E and Fig 3F).

View larger version (99K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Effects of Dmyt1 overexpression in the developing and adult eye. (A) SEM of an eye from a wild-type fly. (B) SEM of an eye from a fly with four copies of GMR-Dmyt1. (C) Eye-antennal disc from a wild-type fly stained with the mitotic marker, antiphosphohistone H3 (PH3), showing mitotic figures in the first (FMW) and second (SMW) mitotic waves. (D) PH3-stained eye-antennal disc from a fly with four copies of GMR-Dmyt1. The SMW is disrupted and delayed, as shown by the decreased number and increased spread of mitotic figures posterior to the FMW. (E) TEM cross section of an adult eye from a wild-type fly. (F) TEM cross section of an adult eye from a fly with four copies of GMR-Dmyt1.

We next tested for genetic interactions with a set of cell cycle regulatory mutants that are predicted to either have a direct regulatory interaction with Dwee1 or Dmyt1 or play an independent role in Cdk1 regulation. Mutations in factors that normally promote the onset of mitosis should enhance the Dwee1 or Dmyt1 overexpression phenotypes, whereas mutations in genes that function to delay mitosis should show the reverse effect. Fig 4 illustrates several such interactions. A single transgene copy of GMR-Dmyt1 produces a mild rough eye phenotype, whereas independently, a heterozygous mutation in cdc25^string has no effect on eye morphology (Fig 4A and Fig B). When a single copy of GMR-Dmyt1 is combined with a heterozygous mutation for cdc25^string, a significantly enhanced eye phenotype is seen (Fig 4C). Likewise, removal of a single copy of cdc2 produces a similar effect in combination with a single copy of GMR-Dmyt1 (Fig 4D). The GMR-Dmyt1/cdc25^string interaction produces an eye that is devoid of bristles, whereas the GMR-Dmyt1/cdc2 interaction shows milder bristle effects. Curiously, the dominant enhancement seen in these cases is consistently stronger in more anterior parts of the eye that differentiate later in development. Cdc2 (now called Cdk1) and its activating phosphatase, Cdc25^string are essential for promoting mitosis in Drosophila (EDGAR and O'FARRELL 1989 ; STERN et al. 1993 ), so these genetic interactions are consistent with known functions for these genes. A weak single-copy GMR-Dwee1 phenotype (Fig 4E) is also enhanced by heterozygous mutant alleles of cdc2 (Fig 4G), but unlike GMR-Dmyt1, not by heterozygous mutations for cdc25^string (not shown). These genetic interactions were confirmed with multiple alleles of cdc2 and cdc25^string to rule out nonspecific genetic background effects. We also tested a number of other known cell cycle mutants for dominant modifier effects on either GMR-Dwee1 or GMR-Dmyt phenotypes. Mutations in cyclin A, cyclin B, mei-41, grapes, twine, cdk2, cyclin E, fizzy, and dacapo all fail to either enhance or suppress the rough eye phenotype generated by either transgene.

View larger version (173K): [in this window] [in a new window] [Download PPT slide]   Figure 4. SEM analysis of eye phenotypes seen in genetic interactions with GMR-Dwee1 and GMR-Dmyt1. (A) SEM showing a fly with a single copy of GMR-Dmyt1. (B) Fly heterozygous for a mutation in the cdc25string locus. (C) Fly with a single copy of GMR-Dmyt1 and heterozygous for a mutation in the cdc25string locus. (D) Fly with a single copy of GMR-Dmyt1 and heterozygous for a mutation in the cdc2 locus. (E) Fly with a single copy of GMR-Dwee1. (F) Fly heterozygous for a mutation in the cdc2 locus. (G) Fly with a single copy of GMR-Dwee1 and heterozygous for a mutation in the cdc2 locus. (H) Fly with a single copy of GMR-rux. (I) Fly with single copies of both GMR-Dmyt1 and GMR-rux. (J) Fly with a single copy of p53-pExP-glass. (K) Fly with single copies of both GMR-Dmyt1 and p53-pExP-glass.

The rux gene encodes a cyclin-dependent kinase inhibitor (CKI) that inhibits Cyclin A/Cdk1 by promoting the degradation of cyclin A (THOMAS et al. 1994 , THOMAS et al. 1997 ; SPRENGER et al. 1997 ; FOLEY et al. 1999 ; AVEDISOV et al. 2000 ). When GMR-Dmyt1 (Fig 4I) or GMR-Dwee1 (not shown) is coexpressed with GMR-roughex (GMR-rux) the phenotype is enhanced relative to that generated by GMR-rux alone (Fig 4H), resulting in a stronger rough eye phenotype that is accompanied by a near complete loss of bristles. While this result is consistent with additive effects of these Cdk1 inhibitors, we also made the surprising observation that otherwise viable zygotic Dwee1 mutants show near-complete synthetic lethality with otherwise viable zygotic rux mutants. Rare double-mutant "escapers" from these genetic crosses show various phenotypic abnormalities, including enhancement of the rux rough-eye phenotype, bristle duplications and deletions, and "Minute" bristles (data not shown).

To investigate genetic interactions with a known component of the DNA damage response pathway, we tested the Drosophila homolog of the p53 tumor suppressor gene. Expression of a p53-pExP-glass transgene promotes apoptosis, generating eye tissue that has no evidence of intact ommatidia or bristles (OLLMANN et al. 2000 ; Fig 4J). Coexpression of a single transgene copy of either GMR-Dmyt1 (Fig 4K) or GMR-Dwee1 (not shown) can markedly suppress this phenotype, with recovery of the eye bristles being most pronounced (compare Fig 4J with 4K).

The tribbles (trbl) gene encodes a novel mitotic inhibitor that functions in mesodermal cells during early gastrulation (GROSSHANS and WIESCHAUS 2000 ; MATA et al. 2000 ; SEHER and LEPTIN 2000 ). ninaE-Gal4-driven UAS-Dwee1 or UAS-trbl transgenes alone generate slightly roughened eyes, with occasional duplication of bristles (Fig 5A and Fig B). When the two genes are coexpressed in the eye, the ommatidial phenotype is dramatically enhanced and there is a near complete loss of bristles (Fig 5C). In a complementary experiment, the eye phenotype generated by two copies of GMR-Dmyt1 combined with a single copy of GMR-Dwee1 is partially suppressed by removal of one gene copy of trbl (data not shown). These striking synergistic interactions are not confined to eye development, as coexpression of UAS-Dwee1 and UAS-trbl yields nearly complete ablation of wing tissue (Fig 5F), compared with scalloping of the wing margin observed when UAS-Dwee1 or UAS-trbl are expressed singly with the sd-Gal4 driver (Fig 5D and Fig E). Occasional conversions of wing tissue to apparent thoracic tissue were also noted in these coexpression experiments. Unlike the similar wing margin phenotypes we observe when UAS-trbl or UAS-Dwee1 are expressed during wing development, UAS-trbl expression is associated with a noticeable reduction of trichome density in the wing blade that apparently reflects increased cell size, a phenotype that is not observed with UAS-Dwee1 (compare Fig 5D and Fig 5E).

View larger version (121K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Coexpression of Dwee1 and trbl shows strong synergistic phenotypic effects. (A) SEM of a fly with one copy of UAS-Dwee1 driven by one copy of ninaE-Gal4. (B) Fly with one copy of UAS-trbl driven by one copy of ninaE-Gal4. (C) Fly with single copies of both UAS-Dwee1 and UAS-trbl driven by a single copy of ninaE-Gal4. (D) Wing of a fly with one copy of UAS-Dwee1 driven by sd-Gal4 (hemizygous on the X chromosome). (E) Wing of a fly with one copy of UAS-trbl driven by sd-Gal4. (F) Fly with single copies of both UAS-Dwee1 and UAS-trbl driven by sd-Gal4. The arrowhead indicates the position of the small piece of wing tissue.

We next conducted genome-wide screens for loci that modify GMR-Dwee1 or GMR-Dmyt1 eye phenotypes, using the Drosophila deficiency kit (maintained by the Bloomington Drosophila Stock Center). The kit presently comprises 195 stocks that are estimated to cover 70–80% of the Drosophila euchromatic genome. In two separate screens, we tested these deletions for their ability to enhance the eye phenotypes associated with single-copy transgenic stocks of either GMR-Dmyt1 or GMR-Dwee1. In a third screen to identify both enhancer and suppressor loci, we tested the deletions against a stock carrying two copies of GMR-Dmyt1 and one copy of GMR-Dwee1 (made by recombination of different transgene insertions). The genetic crosses were scored without reference to whether or not the deletions uncovered any known cell cycle regulators, to avoid biasing our results. The genetic loci that we have identified in these screens, as cytological regions defined either by deletions or by mutations in specific genes, are compiled in Table 1. Consistent with observations based on single alleles, Df(2L)Mdh, which includes the cdc2 locus, enhances the phenotype of all three tester strains, whereas deletions that include cdc25^string [Df(3R)3450 and Df(3R)Dr-rv1] were selected as enhancers of GMR-Dmyt1 and 2xGMR-Dmyt1, 1xGMR-Dwee1 in this assay, but not as enhancers of the GMR-Dwee1 transgene alone.

Six deletions, four of which represent loci not previously identified in crosses with known cell cycle regulators, were identified as specific enhancers of GMR-Dmyt1 (Table 1). One of the GMR-Dmyt1 enhancer regions [Df(3R)Dl-BX12] contains Delta (Dl), which encodes a ligand for signaling through the Notch pathway. Independent tests with specific alleles of Dl have confirmed that Dl is the gene responsible for this interaction. Since some alleles of Dl exhibit dominant eye phenotypes (specifically, Dl¹), it is important to note that we observed enhancement with alleles (Dl³, Dl⁷, Dl^B2, and Dl^RevF10) that by themselves are not associated with a dominant eye phenotype. It is unlikely, therefore, that these interactions reflect additive effects. We saw similar enhancement with gene duplications of the Notch locus, which on their own are associated with a "Confluens" or Delta-like phenotype [Dp(1;2)51b, Dp(1;2;Y)w⁺, and Dp(1;2)72c21]. A deletion of the Notch locus, on the other hand [Df(1)N-8], suppresses the phenotype associated with a 2xGMR-Dmyt1, 1xGMR-Dwee1 strain. Specific genes responsible for the remaining three GMR-Dmyt1 enhancer interactions have not yet been identified. Df(2L)r10 contains three known mitotic regulatory genes (grapes, twine, and fizzy), none of which behaves as an enhancer in tests with specific mutant alleles, however. It is possible that the phenotypic modification seen with this deletion reflects a combinatorial interaction with more than one of these genes.

Only two cytological regions, identified by crosses to the deletion collection, were identified as specific enhancers of a GMR-Dwee1 eye phenotype, one of which contains cdc2 (Table 1). We have not yet identified the gene responsible for the remaining suppressor interaction with 2xGMR-Dmyt1, 1xGMR-Dwee1 that is associated with Df(3L)st4. Further analysis to identify and characterize the remaining gene modifiers will now be necessary to determine if these loci do in fact encode distinct regulators for Dwee1 and Dmyt1.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The G1/S and G2/M cell cycle transitions are temporally and spatially controlled during metazoan development, allowing growth and cell division to be coordinated with patterning and differentiation (reviewed by EDGAR and LEHNER 1996 ). Studies of G2/M checkpoint controls in metazoans have emphasized regulatory mechanisms affecting the Cdc25-like phosphatases, which activate the mitotic regulator Cdk1 by removing inhibitory phosphorylation. Regulatory mechanisms affecting the activity and protein stability of the Cdk1 inhibitory kinases are still poorly understood, but are probably just as important (MICHAEL and NEWPORT 1998 ; LEE et al. 2001 ). There are ample precedents for these mechanisms from studies of Wee1 and Mik1 kinases in S. pombe (RUSSELL and NURSE 1987A ; COLEMAN et al. 1993 ; PARKER et al. 1993 ; WU and RUSSELL 1993 ; O'CONNELL et al. 1997 ; RALEIGH and O'CONNELL 2000 ) and SWE1 in S. cerevisiae (LEW and REED 1995 ; SIA et al. 1996 , SIA et al. 1998 ; BARRAL et al. 1999 ; EDGINGTON et al. 1999 ; MCMILLAN et al. 1999 ).

During the third larval instar, the Drosophila eye disc undergoes progressive transformation from a relatively amorphous epithelial sac into the complex arrangement of ommatidial facets that comprises the adult compound eye. This transformation is marked by passage of a constriction called the MF across the eye disc (WOLFF and READY 1991 ). Cells within the MF normally arrest in G1 and failure to synchronize cells at this stage disrupts ommatidial patterning (THOMAS et al. 1994 ). Following the MF, a population of cells called the SMW undergoes a final cell cycle. If cells are blocked in G1 by overexpression of a p21 CKI homolog, insufficient cells are left to form all of the cell types required for normal ommatidia, resulting in a rough adult eye phenotype (DE NOOIJ and HARIHARAN 1995 ; DE NOOIJ et al. 1996 ). In this report, we have shown that GMR-driven misexpression of Dmyt1 immediately after the MF both delays the SMW divisions and reduces the numbers of mitotic cells, also resulting in a rough eye phenotype.

We have established that Dwee1 and Dmyt1 overexpression eye phenotypes are sensitive to modification by mutations in known cell cycle regulatory genes, illustrating the feasibility of screening for mutations of genes that are potential regulators of either Wee1 or Myt1. Mutations in genes that promote mitosis, such as cdc2 and cdc25^string, should dominantly enhance these overexpression phenotypes and we have confirmed this expectation for both of these genes with Dmyt1. Although a GMR-Dwee1 eye phenotype is also enhanced by mutations in cdc2, it is not enhanced by mutations in cdc25^string, providing evidence that Wee1 and Myt1 kinases have distinct Cdk1 regulatory effects in this developmental context. This result could be explained by a requirement for higher levels of cdc25^string activity to overcome GMR-Dmyt1 inhibition of Cdk1 relative to GMR-Dwee1, perhaps because it is inherently more difficult to dephosphorylate Cdk1 inhibited on both T14 and Y15 by Myt1 activity, compared with Cdk1 inhibited on Y15 alone by Wee1.

The rux gene encodes a novel Cdk1 inhibitor that controls the onset of S phase during embryogenesis, eye development, and spermatogenesis (GONCZY et al. 1994 ; THOMAS et al. 1994 , THOMAS et al. 1997 ; SPRENGER et al. 1997 ; FOLEY et al. 1999 ; AVEDISOV et al. 2000 ). A recent study has shown that rux also plays a novel role in mitosis, by an unknown mechanism (FOLEY and SPRENGER 2001 ). Rux and Wee1 both negatively regulate Cdk1 activity; thus our observation that coexpression of these genes generates more extreme rough eye phenotypes than seen with either alone is consistent with known functions for these genes. Surprisingly, we also found that flies lacking both zygotic Dwee1 and rux functions show nearly complete synthetic lethality, with rare escapers exhibiting extensive adult bristle phenotypes. This interaction suggests that rux and Dwee1 may also cooperate in some other, as yet undefined regulatory mechanism. The extensive bristle phenotypes seen in rux ; Dwee1 double mutant escapers could indicate disruption of cell cycle timing or abrogation of genome integrity checkpoints, similar to the phenotypes seen in mus304 mutants exposed to ionizing radiation, which are associated with increased genome instability (BRODSKY et al. 2000 ). Another piece of evidence suggesting a role for Wee1 kinases in regulating genome stability is the interaction we observe with Drosophila p53. In humans, the p53 tumor suppressor promotes apoptosis in cells that have suffered DNA damage. Overexpression of Drosophila p53 in the eye promotes extensive cell death by apoptosis, resulting in extremely defective eyes (OLLMANN et al. 2000 ). We have shown significant suppression of the p53 overexpression eye phenotype by coexpression of either GMR-Dwee1 or GMR-Dmyt1, suggesting that these Cdk1 inhibitory kinases can negatively regulate p53-induced apoptosis. Since Cdk1 activity has previously been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition (ZHOU et al. 1998 ). Other reports relevant to this issue are somewhat contradictory, however. In human cell culture, Wee1 can inhibit granzyme B-induced apoptosis; furthermore, Wee1 appears to be downregulated through a p53-dependent mechanism, suggesting that p53 regulation of Wee1 might normally occur during this process (CHEN et al. 1995 ; LEACH et al. 1998 ). In contrast, SMITH et al. 2000 showed that Wee1 activity can actually promote apoptosis in a Xenopus oocyte extract system. Further studies are clearly needed to establish the physiological significance of any purported roles for Wee1 or Myt1 in regulating apoptosis, p53-dependent or otherwise.

A screen for modulators of wee1 overexpression was previously conducted in S. pombe, by isolating suppressors of wee1-induced lethality (ALIGUE et al. 1994 ; MUNOZ and JIMENEZ 1999 ; MUNOZ et al. 1999 ). These studies identified mutations in the gene encoding the Hsp90 chaperone as potent suppressors, suggesting a role for Hsp90 in promoting the assembly and/or disassembly of functional Wee1 protein complexes. In contrast, we have not found hsp83 mutant alleles (encoding Drosophila Hsp90) to act as suppressors of a combined GMR-Dmyt1/GMR-Dwee1 transgene eye phenotype (data not shown). We have, however, identified several other genetic loci as specific enhancers of eye phenotypes generated by GMR-Dwee1 or GMR-Dmyt1 alone, indicating that phenotypic effects mediated by Wee1 and Myt1 are responsive to lowered expression of different genes. These observations may reflect differences in threshold requirements for the relevant gene products in promoting mitosis (as suggested by the interactions with cdc25^string) or they may signify differences in the regulation of Wee1 and Myt1 kinases that we will now be able to dissect by identifying and characterizing the relevant modifier loci. We are currently undertaking direct genetic screens for mutations in genes that modify GMR-Dwee1 and GMR-Myt1 eye phenotypes to address this issue. One of the loci we have identified as a specific enhancer of the GMR-Dmyt1 eye phenotype is Delta. This interaction could reflect defects in Dl-dependent neuronal specification that are enhanced by GMR-Dmyt1 activity, or it may indicate a novel role for Delta/Notch signaling in regulating Myt1 activity. We are presently trying to distinguish these possibilities.

In S. pombe, the DNA damage and DNA replication checkpoint pathways that regulate Cdk1 by inhibitory phosphorylation act by controlling the activity and stability of Wee1 and Mik1 kinases, as well as Cdc25 phosphatases (reviewed by WALWORTH 2000 ). Although metazoan homologs of components of these checkpoint pathways show significant sequence conservation with their yeast homologs, the actual functions and interactions of individual components are not necessarily conserved. For example, GUO and DUNPHY 2000 showed that Xenopus homologs of the checkpoint kinases Chk1 and Cds1, which respond to DNA damage and blocked DNA replication, respectively, in S. pombe, respond in the exact opposite manner to these stresses in Xenopus egg extracts. This example serves as a warning that simple predictions of metazoan gene function based on extrapolation from known functions of yeast genes can be misleading. Metazoan development requires that novel regulatory mechanisms exist to link specific developmental processes with the basic cell cycle machinery. Drosophila represents an ideal model for analyzing these developmental controls of the cell cycle, since the effects of specific mutations on complex processes like morphogenesis and differentiation can be established. The recent characterization of the trbl gene in Drosophila illustrates this point (GROSSHANS and WIESCHAUS 2000 ; MATA et al. 2000 ; SEHER and LEPTIN 2000 ). Trbl activity delays mitosis in invaginating G2 cells (mitotic domain 10) in a cycle 14 embryo. Although cdc25^string transcription initiates in domain 10 before it is transcribed in other cells, these cells remain G2 arrested until they are completely internalized, well after cells in nine other mitotic domains have subsequently expressed cdc25^string and entered mitosis (EDGAR and O'FARRELL 1989 ). Trbl activity downregulates Cdc25^string protein stability, providing an explanation for these observations (MATA et al. 2000 ). A similar purpose could be served by Trbl simultaneously upregulating Dwee1 or Dmyt1 activity (GROSSHANS and WIESCHAUS 2000 ). Intriguingly, Trbl contains motifs reminiscent of Nim1-type kinases, which negatively regulate Wee1 and Swe1 kinase activity and stability in S. pombe and S. cerevisiae (RUSSELL and NURSE 1987A ; COLEMAN et al. 1993 ; PARKER et al. 1993 ; WU and RUSSELL 1993 ; BARRAL et al. 1999 ). Despite these sequence similarities, the Trbl protein apparently lacks a functional catalytic domain, raising the possibility that Trbl could act in a "dominant negative" manner to activate Wee1 (or Myt1) by interfering with the activities of Nim1-like inhibitors. Genetic interactions that we describe in this report are consistent with this possibility.

	ACKNOWLEDGMENTS

We thank Rakesh Bhatnagar and George Braybrook for assistance with electron and confocal microscopy, Christine Walker for assembling the pUASp-Dwee1 clone and assisting with embryo injections, Scott Hanna for advice and assistance in making transgenic flies, Bruce Hay for the gift of GMR plasmid, Gary Ritzel for advice on synthesizing the GMR clones, Veronica Rodrigues for the sd-Gal4 stock, Barbara Thomas for the GMR-rux stock, J. Grosshans for the UAS-trbl stock, and Exelixis for providing the p53-pExP-glass stock. The Bloomington Drosophila Stock Center provided other stocks described in this work. Funding was provided by research grants from the Alberta Heritage Foundation for Medical Research (AHFMR) and the Canadian Institutes of Health Research to S.D.C. and by graduate student fellowships from the Natural Sciences and Engineering Research Council of Canada and AHFMR to D.M.P.

Manuscript received July 9, 2001; Accepted for publication March 11, 2002.

	LITERATURE CITED

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