Digoxygenin-labelled Wee mRNA antisense probes were hybridized to Drosophila embryos to study expression of the gene during early development. Abundant maternal Wee mRNA is seen throughout the embryo during the early rapid nuclear cycles. This mRNA is degraded early in interphase 14, at a time when many maternal mRNAs (including String) are degraded. However, maternal Wee mRNA persists in the pole cells and shows a perinuclear, granular signal similar to that seen with perduring maternal cyclin B mRNA in pole cells during the same stage. Low levels of Wee mRNA reaccumulate throughout the embryo during gastrulation, indicating that zygotic transcription of the gene is activated at this time. This zygotic expression is fairly uniform (although consistently stronger in the mesoderm) and has no discernible pattern related to cell cycle progression during the three major post-blastoderm divisions. After germ band retraction, Wee expression is observed in the mitotically active nervous system, as well as in hindgut, foregut and midgut tissues that have withdrawn from proliferative cell cycles and are beginning to endoreplicate. Still later in embryogenesis, the proliferating cells of the central nervous system express high levels of Wee mRNA, while expression is substantially lower in the rest of the embryo. Strong Wee expression is also seen in the brain of a first instar larva (Campbell, 1995).
Deletion mutants of wee were generated by transposase-mediated imprecise excision of a nearby P{w+} transposon insertion, associated with l(2)k10413. Complementation tests have shown that all three female-sterile mutations recovered (Dwee1ES1, Dwee1ES2, and Dwee1DS1) are alleles of the same gene, and this gene corresponds to Dwee1. A detailed phenotypic analysis was undertaken of one of the mutant alleles, Dwee1ES1. Hemizygous Dwee1ES1 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. This phenotype and the subsequent clumping and fragmentation of nuclei observed is identical to what is seen in embryos collected from grp or mei-41 mutant females. This observation suggests a possible role for Dwee1 in the same developmental checkpoint as mei-41 and grp (Price, 2000).
Two different approaches were undertaken to demonstrate that the complementation group represented by the three female-sterile mutations does in fact correspond to wee. (1) genomic DNA isolated from adults hemizygous for each of the alleles was sequenced. With respect to their maternal phenotype, Dwee1ES1 and Dwee1ES2 behave as classical amorphic alleles. Genomic DNA isolated from each of these mutants contains a DNA lesion within the kinase domain of wee that is expected to either abolish or severely disrupt the function of the gene. Dwee1ES1 contains an 8-bp deletion causing a frameshift followed by a stop codon, truncating the protein in kinase domain IV. Dwee1ES2 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). Dwee1DS1 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 has shown 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 Dwee1ES1 allele shows an antimorphic interaction with the Dwee1DS1 allele in that the phenotype of embryos derived from Dwee1DS1/Dwee1ES1 transheterozygous mothers is more severe (embryos never cellularize) than seen in Dwee1DS1/Df(2L)Dwee1WO5 hemizygotes. Conceivably, this reflects titration of positive regulatory factors by the truncated Dwee1ES1 protein, thus lowering the effective levels of Dwee1DS1 function (Price, 2000).
(2) The phenotype of mutant embryos can be partially rescued with a heat-inducible wee cDNA transgene. Maternal Dwee1ES1 hemizygous flies carrying this transgene were briefly heat-shocked to induce expression as confirmed by immunoblot analysis. 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. Wide phenotypic variation was observed in the extent of phenotypic rescue, presumably reflecting variations in the amount and timing of Wee 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. A single transgene copy of a genomic DNA construct that contains wee 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 wee are found in the female-sterile mutants and also show that wee expression is both necessary and sufficient to rescue the maternal lethal phenotype. It is concluded from this evidence that mutant alleles of wee have been isolated. The striking similarity between the phenotype of wee mutant-derived embryos and embryos derived from grp or mei-41 mutants provides a strong argument that maternally provided wee plays an essential role in the same developmental process as grp and mei-41 (Price, 2000).
Additional evidence in favor of this hypothesis is afforded by providing extra maternal copies of the genomic wee transgene in a mei-41D3 mutant background. Females homozygous for the mei-41D3 allele produce cellularized embyros at a very low frequency (2%). The frequency of cellularized embryos is dramatically increased by adding an extra maternal copy of a wee genomic transgene (20%). The mei-41D3 mutant embryos are further rescued by addition of two wee transgenes (50%), to the extent that some mei-41D3-derived embryos are 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 wee. The simplest interpretation that can be offered for why the results differ between grp and mei-41 mutants in these experiments is that the mei-41D3 is not a complete loss-of-function allele, and consequently mei-41D3 mutants are more sensitive to increased dosage of wee than grp1 mutants. Alternatively, grp may respond to two different signaling pathways whereas mei-41 may respond to only one of the two. Wee1 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 Dwee1DS1 maternal background. The incompletely penetrant syncytial arrest phenotype of homozygous Dwee1DS1-derived embryos (54% cellularized) was enhanced by subtracting a maternal copy of mei-41+ (39%). Removal of one maternal copy of grp+ produced an even greater enhancement of the mutant phenotype of Dwee1DS1 embryos (29% cellularized) (Price, 2000).
It was of interest to assess whether wee hemizygous flies derived from heterozygous parents would be 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. For this experiment, the sensitivity was assessed of Dwee1ES1 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. Genetic crosses between balanced heterozygous stocks carrying either the Dwee1ES1 mutant chromosome or the Df(2L)Dwee1W05 chromosome generate both heterozygous and hemizygous viable adult progeny. Exposure to 1 or 2 mM HU eliminates the hemizygous Dwee1ES1 class of progeny, indicating that wee mutant larvae are indeed highly sensitive to HU, presumably reflecting a requirement for wee activity in a fully functional DNA replication checkpoint (Price, 2000).
Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. This study presents the identification of Drosophila Myt1 kinase and examines the relationship of Myt1 and Wee activities in the context of Cdc2 phosphorylation. Myt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells results in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) results in an increased rate of proliferation. In addition, loss of Myt1 alone or in combination with Drosophila Wee1 (Wee1) results in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of Myt1 alone results in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of Cdc2 on the tyrosine-15 (Tyr-15) residue is only observed when expression of both Myt1 and Wee expression is reduced via RNAi and not loss of expression of Wee alone. Most strikingly, in the absence of Myt1, Golgi fragmentation during mitosis is incomplete. These findings suggest that Myt1 and Wee have distinct roles in the regulation of Cdc2 phosphorylation and the regulation of mitotic events (Cornwell, 2002).
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). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used 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 these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified 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. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).
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. 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. There are ample precedents for these mechanisms from studies of Wee1 and Mik1 kinases in S. pombe and SWE1 in S. cerevisiae (Price, 2002).
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 morphogenetic furrow (MF) across the eye disc. Cells within the MF normally arrest in G1 and failure to synchronize cells at this stage disrupts ommatidial patterning. Following the MF, a population of cells called the second mitotic wave (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. In this report, 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 (Price, 2002).
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 cdc25string, should dominantly enhance these overexpression phenotypes and this expectation was confirmed 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 cdc25string, 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 cdc25string 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 (Price, 2002).
The rux gene encodes a novel Cdk1 inhibitor that controls the onset of S phase during embryogenesis, eye development, and spermatogenesis. rux also plays a novel role in mitosis, by an unknown mechanism. Rux and Wee1 both negatively regulate Cdk1 activity; thus the 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, 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. Another piece of evidence suggesting a role for Wee1 kinases in regulating genome stability is the interaction observed 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. There is 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 been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition. 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. In contrast, 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 (Price, 2002).
A screen for modulators of wee1 overexpression has been conducted in S. pombe, by isolating suppressors of wee1-induced lethality. 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, no hsp83 mutant alleles (encoding Drosophila Hsp90) were found to act as suppressors of a combined GMR-Dmyt1/GMR-Dwee1 transgene eye phenotype. Several other genetic loci have been identified 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 cdc25string) or they may signify differences in the regulation of Wee1 and Myt1 kinases that it will now be possible to dissect by identifying and characterizing the relevant modifier loci. Direct genetic screens to address this issue are being undertaken for mutations in genes that modify GMR-Dwee1 and GMR-Myt1 eye phenotypes. One of the loci 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 (Price, 2002).
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. 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, Xenopus homologs of the checkpoint kinases Chk1 and Cds1, which respond to DNA damage and block 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 tribbles gene in Drosophila illustrates this point. Trbl activity delays mitosis in invaginating G2 cells (mitotic domain 10) in a cycle 14 embryo. Although cdc25string 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 cdc25string and entered mitosis. Trbl activity downregulates Cdc25string protein stability, providing an explanation for these observations. A similar purpose could be served by Trbl simultaneously upregulating Dwee1 or Dmyt1 activity. 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. 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 described in this study are consistent with this possibility (Price, 2002).
During Drosophila oogenesis, unrepaired double-strand DNA breaks activate a mei-41-dependent meiotic checkpoint, which couples the progression through meiosis to specific developmental processes. This checkpoint affects the accumulation of Gurken protein, a transforming growth factor alpha-like signaling molecule, as well as the morphology of the oocyte nucleus. However, the components of this checkpoint in flies have not been completely elucidated. A mutation in the Drosophila Chk2 homolog (DmChk2/Mnk or loki) has been shown to suppress the defects in the translation of gurken mRNA and also the defects in oocyte nuclear morphology. Drosophila Chk2 is phosphorylated in a mei-41-dependent pathway. Analysis of the meiotic cell cycle progression shows that the Drosophila Chk2 homolog is not required during early meiotic prophase, as has been observed for Chk2 in C. elegans. The activation of the meiotic checkpoint affects Wee localization and is associated with Chk2-dependent posttranslational modification of Wee. It is suggested that Wee has a role in the meiotic checkpoint that regulates the meiotic cell cycle, but not the translation of gurken mRNA. In addition, p53 and mus304, the Drosophila ATR-IP homolog, are not required for the patterning defects caused by the meiotic DNA repair mutations. It is concluded that Chk2 is a transducer of the meiotic checkpoint in flies that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 in this specific checkpoint affects a cell cycle regulator as well as mRNA translation (Abdu, 2002).
In the budding yeast, checkpoint-dependent cell cycle arrest at pachytene is achieved by the accumulation of hyperphosphorylated Swe1p, a Wee1-like protein, and subsequent inactivation of Cdc28p. Like other metazoans, Drosophila has two Wee1-like kinases, Wee and Dmyt1. To study the role of Wee in the meiotic checkpoint, the Wee expression in ovaries from spindle-class mutants was compared to expression in wild-type by using an anti-Wee antibody. Western blot analysis shows that the mobility of Wee1 protein is retarded in spn-B, okr, and spn-D mutant ovaries. Wee1 protein also migrates slowly in ovarian extracts prepared from flies mutant for spn-B and grp. In contrast, the mobility of Wee in flies mutant for spn-B and Chk2 is restored to wild-type. Immunohistochemical assays also show an abnormal Wee subcellular localization in spindle-class genes. In wild-type ovaries, Wee protein accumulates inside the oocyte nucleus but is excluded from the DNA, whereas, in about 37% of mutant egg chambers from spn-B, okra, and spn-D, Wee protein accumulates throughout the oocyte nucleus. Interestingly, it was found that mutations in Wee are not able to suppress the dorsal-ventral patterning or the oocyte nuclear morphology defects caused by mutations in the spindle-class genes. Expression of an active form of Cdc2 alone or together with Cyclin A in spn-B mutant flies does not suppress these defects (Abdu, 2002).
The changes in the Wee expression in spindle-class mutants suggest that the initiation of the meiotic checkpoint affects the meiotic cell cycle progression in a Wee-dependent manner, as it does in yeast. However, mutations in Wee1 do not suppress the patterning defects in spindle-class mutants. It is possible that two different pathways are activated by the persistence of unrepaired double-strand DNA breaks, one affecting Wee and the cell cycle, and a second pathway leading to Vasa modification and patterning defects. Alternatively, it is possible that other cell cycle regulators act in parallel to Wee and that the primary effect of the checkpoint is cell cycle arrest, which in turn affects Vasa modification and patterning. However, several studies suggest that, in spindle mutants, there is only a transient cell cycle arrest during early oogenesis, whereas the major effect on translation of grk mRNA occurs during mid-oogenesis. Thus, it is proposed that the patterning defects in spindle mutants are not the result of checkpoint-induced cell cycle arrest (Abdu, 2002).
In summary, the results demonstrate that the Drosophila Chk2 homolog is a transducer of the meiotic checkpoint that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 results in modification of two proteins, Vasa and Wee, which then affect progression of the meiotic cell cycle and translation of gurken mRNA. Wee is, however, not required for the patterning defects seen in the spindle mutations. Activation of the Chk2-dependent meiotic checkpoint may therefore control several cell cycle regulators which in turn may affect both meiosis and translation of gurken mRNA. In particular, it is likely that Wee1 activation regulates cell cycle progression, whereas Chk2 may utilize an independent target to regulate Vasa, which subsequently affects dorsal-ventral patterning as well as nuclear morphology of the oocyte. While dorsal-ventral signaling by Gurken is not a conserved feature of oogenesis found in other organisms, the fact that homologs of Drosophila Chk2 act during meiosis in other organisms raises the possibility that meiotic checkpoints in other species might also act through Chk2 to regulate translation during oogenesis and thus directly link the meiotic cell cycle to the development of the oocyte (Abdu, 2002).
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date revised: 15 February 2015
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