The genetic analysis of png suggests that it is a cell cycle regulator specific for early embryogenesis, because all of the mutations, including those predicted to ablate kinase activity, are strict maternal effect lethal alleles. Thus, the gene is not required for viability at any point in development beyond the maternal requirements in early embryogenesis. It is plausible that there are regulators specific for the early division cycles. The early embryonic S-M cell cycle has a pattern in which DNA replication and mitosis oscillate rapidly without gap phases. This regulation must be controlled by maternal products in the egg, since zygotic transcription does not occur until the tenth division of embryogenesis. In addition to being regulated post-transcriptionally, this cycle also differs from the archetypical cell cycle in that it occurs in the presence of large maternal pools of proteins such as cyclins (Fenger, 2000).
To examine whether png expression is specific for early embryogenesis, a Northern blot of polyA+ RNA isolated from different developmental stages was probed with the png ORF. Only a single transcript of 1.3 kb was detected, and it was expressed predominantly in adult females and early embryos until 3 hours of embryogenesis, consistent with it being maternally deposited in the egg. Barely detectable levels of the transcript are present in embryos between 3 and 6 hours, suggesting that the transcript is degraded or no longer expressed after the mid-blastula transition. png transcript was also present in the Schneider L2 embryonic cell line (Fenger, 2000).
To examine Png protein levels throughout development, polyclonal antibodies were generated against a fusion protein consisting of GST fused to the C-terminal 167 amino acids of Png. An immunoblot of extracts from different developmental stages was probed with anti-Png antibodies. Wild-type Png protein is highly expressed in unfertilized eggs and in 0 to 2 hour embryos, which is consistent with the timing of the defect in png mutants. Png is present at low levels until 6 to 12 hours of embryogenesis. This could be due to persistence of the high levels of maternal protein, or it could be from the presence of unfertilized eggs in the collections. Png protein is not present in 12 to 24 hour embryos, nor is it expressed in the endoreplicating larval salivary glands, mitotic larval brains or the imaginal discs that form the adult tissues (Fenger, 2000).
Mutations in the Drosophila maternal genes plutonium and pan gu have the striking phenotype that DNA replication initiates in unfertilized eggs. Fertilized eggs from plu or png mutant mothers also have a mutant phenotype: DNA replication is uncoupled from nuclear division, resulting in giant, polyploid nuclei. Analysis of multiple alleles of these genes indicates that their wild-type function is required to maintain repression of DNA replication until fertilization. The phenotype of two png alleles suggests that this gene also may play a direct role in coupling S phase and mitosis during the early cleavage divisions. Genetic interactions among png, plu, and the previously identified gene gnu are described that demonstrate these three genes regulate the same process (Shamanski, 1991).
The existing png alleles fall into two phenotypic classes. All of the mutations cause inappropriate replication of the meiotic products in unfertilized eggs. The embryonic phenotype differs in the alleles, however. In three of the alleles, no mitosis occurs; the three polar bodies and two pronuclei replicate and fuse into a multi-lobed giant nucleus. In two of the alleles, some mitotic divisions occur, but ultimately the nuclei undergo solely DNA replication to become polyploid. It is postulated that the transient S-M linkage that occurs in some png alleles to produce embryos with multiple nuclei is due to partial function (Shamanski, 1991). In order to confirm this, it was thought useful to examine more alleles of png and determine if other weak alleles had the same multinuclear phenotype or a different phenotype. The laboratory of Beat Suter provided four new maternal-effect lethal mutations with a giant nuclei phenotype. These four X-linked EMS mutations failed to complement the female sterility of png1058 and png3318, indicating that they are new alleles of png. To examine if any of the new png alleles had a different phenotype from the previously isolated alleles, fixed embryos from homozygous mutant mothers were stained with propidium iodide to visualize the DNA. All had giant polyploid nuclei, but fell into two classes: png172 and png246 show no evidence of mitosis, whereas embryos from png50 and png171a undergo a few mitoses before the nuclei over-replicate. One allele, png50, is different in that the embryos undergo more mitoses than had been seen with other png alleles. Embryos from png50 mothers have up to 64 nuclei, indicating that they complete one or two more divisions than png3318 or png171a, the other alleles that have transient S-M cycles. The two new multinuclear alleles confirm that this phenotype is due to loss of png function, and if the strength of the allele is inversely proportional to the number of nuclei, then png50 is the weakest of the eight alleles (Fenger, 2000).
It was of interest to determine the important amino acids in the Png protein, and to test if the nature of the mutations was consistent with the strength of the allele phenotypes. Molecular analysis of the mutations enabled a determinination of important protein regions and facilitated an understanding of the nature of the two classes of alleles. The png ORF was amplified from homozygous mutants using PCR and the PCR products were sequenced. The sequence reveals that all alleles cause changes in the kinase domain. Four of the mutations with severe phenotypes (the strong alleles) are predicted to have pronounced effects on kinase activity: (1) png172 is a missense mutation that causes a glycine-to-glutamate change in the DFG loop of the kinase domain. The DFG loop is required for primary Mg 2+ chelation, which helps orient the gamma-phosphate for transfer, and the glycine residue is invariant in almost all protein kinases. (2) png1920 causes a proline-to-serine change at residue 140 in the conserved catalytic loop. Pro140 itself is not absolutely conserved in protein kinases, but it is surrounded by critical residues at the active site. It is near Asp137, considered to be the catalytic base that accepts a proton from the attacking hydroxyl group of the substrate. It is also near Asn142, which stabilizes the catalytic loop by hydrogen bonding to the backbone carbonyl of Asp137, and chelates the secondary Mg 2+ ion that bridges the alpha- and gamma-phosphates of the ATP. Finally, it is next to invariant residue Lys139, which is thought to neutralize the negative charge of the gamma-phosphate during transfer. (3) The mutation in png1058 predicts an arginine-to-cysteine change at residue 265 near the C terminus. Arg265 is invariant in all kinases and is important for the kinase structure because it forms an ion pair with Glu181. (4) png158 is a nonsense mutation that truncates the last 30 amino acids of the protein and also removes the invariant Arg265. The severity of the png158 mutation, and the alterations of conserved regions in png172, png1920 and png1058 are consistent with these alleles causing severe phenotypes (Fenger, 2000).
In contrast, the amino acid changes of the mutations that give a weaker phenotype, permitting a few mitotic divisions, would be predicted to result in only partial loss of kinase activity. The weak png3318 and png2786 alleles have the same glycine-to-serine mutation, which alters an invariant residue in the glycine loop required for ATP binding. This glycine-to-serine change is a neutral change, so the kinase could still be partially functional. The mutations of png50 and png171a both alter residues that are not conserved in protein kinases, consistent with these alleles being weak loss-of-function alleles. In summary, the absence of mitosis in embryos from mutant mothers correlates with mutations in png predicted to eliminate or severely reduce kinase activity, whereas mutations that should have diminished kinase activity produce a phenotype in which mitosis occurs for the first few embryonic divisions (Fenger, 2000).
If png were required throughout development, then a null allele would be lethal. Since all of the alleles are homozygous viable, it was of interest to determine whether any were null mutations. The classic genetic test for leaky alleles, comparing the phenotype of homozygous mutants with a mutation in trans to a deficiency, could not be applied to png mutants. For both classes of png alleles, homozygous female mutants have the same phenotype as females with alleles in trans to a deficiency, presumably because of high maternal expression (Shamanski, 1991). Whether the Png protein is missing in any of the alleles was examined by doing immunoblots of 0-1 hour embryo extracts from wild-type or homozygous mutant mothers. None of the alleles was a protein null, and Png protein levels in the eight mutants were only slightly decreased compared with wild-type levels. Although none of the png mutants is lacking protein, and thus are not proven to be null, the png172 allele is likely to be null because it alters a conserved amino acid of the kinase domain. As a final test to determine whether Png could regulate an archetypical cell cycle, png was ectopically expressed in the eye imaginal disc during eye morphogenesis. Wild-type Png was expressed under the control of Glass using the pGMR vector. pGMR-PNG transformant flies had no apparent eye defects. To test if Png required Plu to affect eye development, flies expressing both Plu and Png in the eye imaginal discs were examined. Animals carrying both pGMR-png and a pGMR-plu transgene known to express Plu (Elfring, 1997) had completely wild-type eyes. Png and Plu were also expressed under the control of the inducible hsp70 promoter. Heat-shock induction of Png and Plu either during embryogenesis or larval development did not affect recovery of viable, phenotypically normal adults (Fenger, 2000).
A genetic screen was initiated to identify mutants that enhance or suppress weak png mutations by using the deficiency collection available from the Bloomington Stock Center to survey about 50% of the genome using a minimum number of stocks and crosses. Deficiencies that when heterozygous would dominantly suppress or enhance weak png mutations were scored by examining the nuclear phenotype of embryos. Females transheterozygous for the weak png3318 allele and the strong png1058 allele produce embryos in which some mitotic divisions occur before the nuclei ultimately become polyploid. These embryos contain up to 16 giant, polyploid nuclei. In the same collections, there are embryos that have a single, multilobed nucleus that results from the female meiotic products and male pronucleus undergoing DNA replication but no nuclear division. As these nuclei become polyploid they can fuse together. Thus, in the absence of mitosis and nuclear division, between one and five giant polyploid nuclei are produced. Two classes of embryos were scored as those with five or fewer nuclei vs. those with greater than five (multinucleated). The relative percentages of these two classes produced by png3318/png1058 females is affected by genetic background and can vary between 30% and 55%. Thus suppression was defined as genotypes producing >60% multinucleated embryos and enhancement as <30% (Lee, 2001).
Embryos were collected for 0–4 hr from y png3318 w/y png1058 w females carrying a deficiency on the second or third chromosome. The females with the deficiency were distinguished from their sibling controls by the absence of a dominant P[w+] marker carried on the autosome. Enhancement of the png mutant phenotype was evident by a reduction in the number of embryos with greater than five nuclei. Suppression was identified by the appearance of an increased number of embryos with multiple nuclei; strong suppressors also produced some embryos that had a greater number of nuclei than the maximum of 16 typically seen in embryos from png3318/png1058 females. Some of the deficiencies that were identified as suppressors were subsequently tested for their ability to suppress females homozygous for the strong allele, png1058/png1058 to produce embryos with multiple nuclei (Lee, 2001).
59 deficiency intervals on the second chromosome and 42 intervals on the third chromosome were screened. These deficiencies spanned ~62% of the second and ~47% of the third chromosome. As a control for the screen's effectivness, a deficiency was recovered for plu (Df(2R)173) as an enhancer of the png phenotype. Eight deficiencies were recovered that strongly enhance the png phenotype, 12 that weakly enhance, and 8 that suppress png mutations (Lee, 2001).
To identify the locus within each deficiency responsible for the genetic interaction with png, lethal P-element insertions within or adjacent to each interval were tested for interaction with png and, when possible, deficiencies that dissect the interval were examined. This screen led to the identification of five single complementation groups that interact genetically with png. The strongest enhancer identified was cyclin B. The strongest suppressors were eIF-5A and PP1 87B. Two weaker enhancers were recovered: l(2)s4989 is a P-element insertion that disrupts a novel gene and l(2)10481 is a P-element insertion 5' to the Tkr gene (Tyrosine kinase-related) (Lee, 2001).
Two deficiencies that remove the 59AB region on chromosome 2R strongly enhanced the pan gu mutant phenotype. This region contains the cyclin B gene, a strong candidate for the interacting locus given the observation that Cyclin B protein levels are decreased in png mutants in proportion to the strength of the allele. Further reduction of Cyclin B protein by mutation of one copy of the gene would be predicted to enhance the png phenotype. To test whether the enhancement was due to a decreased gene dosage of Cyclin B, an allele of cyclin B was obtained that is a small deletion generated by imprecise excision of a P element. This mutation strongly enhances the png phenotype (Lee, 2001).
Conversely, it was of interest to test whether the phenotype could be suppressed by increasing the level of Cyclin B protein in the png mutant embryos, so multiple copies (six to eight) of the wild-type cyclin B gene were crossed into the png mutant background. Strains with extra copies of the cyclin B gene in the png mutant background produce increased amounts of Cyclin B protein. Overexpression of cyclin B strongly suppresses the png mutants. In the weak png3318 mutants with extra copies of the cyclin B gene, there is a striking increase in the number of nuclei in the embryos as well as in the number of embryos with multiple nuclei. In addition, the polar bodies contain condensed chromosomes with a normal rosette arrangement. This extent of suppression was not observed with any other deficiency or mutant tested. Moreover, the chromosomes of the zygotic nuclei were condensed, and mitotic figures were visible. The suppression of the zygotic nuclei was not complete, however, because all of the embryos contained polyploid nuclei, and none survived past embryogenesis. Thus, the linkage between S and M phases was ultimately broken, and nuclear division ceased. Overexpression of cyclin B dramatically suppresses the strong png1058 mutants as well, resulting in multiple nuclei in these embryos. However, unlike the weak png3318 mutants with extra cyclin B, normal polar bodies were not observed (Lee, 2001).
Cyclin B levels were also increased in png mutants by heat-shock induction and by induction in the germline using GAL4 under the control of the nanos promoter. The cyclin B gene in the latter construct lacks the N-terminal destruction box and thus would promote elevated levels of Cyclin B both from transcriptional induction as well as lack of degradation by the ubiquitin-APC/C pathway. Both the weak and strong png phenotypes are suppressed by overexpression of cyclin B by either of these two methods. However, the degree of suppression of the zygotic nuclear phenotype of png is less than that for lines carrying extra copies of the wild-type cyclin B gene, and restoration of normal polar body morphology was not observed (Lee, 2001).
Given the observation that png, plu, and gnu appear to function in a common pathway, the genetic interactions between png and cyclin B, and the decreased Cyclin B protein levels in plu and gnu mutants, it was of interest to test whether or not the plu and gnu phenotypes could be modified by altering the cyclin B gene dosage. Unlike png, all of the existing mutations in plu and the single gnu mutation appear to be null alleles. Because mitosis occurs only very rarely in embryos from plu and gnu mutant females, it is not possible to identify enhancers of this strong phenotype. Deficiencies identified in this screen as suppressors of the png phenotype did not suppress the plu phenotype, perhaps due to a lack of residual Plu function. Surprisingly, increasing the level of Cyclin B protein in plu6 and gnu305 mutants by increasing the cyclin B gene dosage (four extra copies) results in suppression of the mutant phenotypes. For plu females with extra cyclin B, 39% of their embryos were multinucleated compared to 1.5% in sibling controls; for gnu females with extra cyclin B, 65% of their embryos were multinucleated compared to 0.8% in sibling controls. Similar results were obtained by inducing nondegradable Cyclin B in the germline of the plu mutant. Unlike the weak png mutation, overexpression of cyclin B does not correct the polar body defects associated with plu and gnu mutations (Lee, 2001).
In addition to Cyclin B, the levels of Cyclin A protein are decreased in proportion to the strength of the png allele. Surprisingly, neither a deficiency that uncovers cyclin A nor mutations in the gene enhance the png mutant phenotype. A negative result in this test does not exclude the possibility of interaction because a twofold reduction in gene dosage may not decrease the level of gene product below a threshold required to detect an effect. However, loss of one copy of the cyclin A gene in the mother does affect cell cycle parameters and nuclear division in the embryo, so one copy of the gene does not produce sufficient levels of the protein for normal cell division. Whether overexpression of cyclin A could suppress the png mutant phenotype was tested. This experiment produced an unexpected result. When cyclin A was induced by heat shock, the png phenotype was enhanced. This may be due to the ability of Cyclin A when overexpressed to drive cells inappropriately into S phase. Thus, this result may be a consequence of artifactually high levels of Cyclin A and may not accurately reflect the function of Cyclin A in the early embryonic divisions (Lee, 2001).
Mutation of the cyclin B3 gene has no effect on the png phenotype, but crossing eight extra copies of the wild-type cyclin B3 gene into the png mutant background results in suppression. A deficiency that uncovers cyclin J does not modify the png mutant phenotype. The cyclin E gene is required for S phase in Drosophila. Whether a deficiency that uncovers cyclin E or a mutation in the cyclin E gene would dominantly suppress png was tested. Unexpectedly, it was found that a decreased gene dosage of cyclin E results in strong enhancement of the png phenotype. As an independent test for potential interactions between cyclin E and png, a mutation in dacapo, an inhibitor of the CDK2/Cyclin E kinase was tested. Both the dacapo mutation and a deficiency that uncovers dacapo (Df(2R)B5) slightly suppress the png mutant phenotype (Lee, 2001).
The deficiency Df(2R)Px2 strongly suppresses the png mutant phenotype. To identify the gene responsible for this interaction, available lethal P element insertions both within the genomic interval of this deficiency and in immediately adjacent-lettered divisions were tested. A P-element inserted within the first intron of the eIF-5A gene was found to suppress png. The eIF-5A mutation lies in the region of Df(2R)Px2, but it complements the deficiency and thus lies outside of it. Consequently, both eIF-5A and another as yet unidentified locus inside the deficiency are suppressors of png. That the eIF-5A mutation is a suppressor was confirmed by testing an independent P-element insertion in the eIF-5A gene. The lethal phenotype of these mutations has not been analyzed, and the gene is defined on the basis of its homology to eukaryotic orthologs of the eIF-5A protein. Although the eIF-5A protein was identified by its ability to promote peptide bond formation in vitro, its role in translation and gene expression is not well understood. It may contribute to RNA stability rather than to translation initiation. No increase in Cyclin B protein levels could be detected in embryos from png1058/png1058; eIF-5A/+ females by immunoblotting (Lee, 2001).
To test whether the effect of eIF-5A on png could be due to perturbations in translation initiation, mutations in the translation initiation factors eIF-4a, eIF-4E, and eIF-3p40 were crossed into png mutants, but no enhancement or suppression of the phenotype was observed. Mutations in two other genes known to be involved in translation, poney and plume were examined; they did not genetically interact with png. Although these results do not eliminate the possibility of a link between translation levels and the png phenotype, they suggest that the effect of eIF-5A is not due to a global decrease in translation (Lee, 2001).
Reduction of the gene dosage of the type 1 serine/threonine protein phosphatase PP1 87B appears to be responsible for suppression of the png phenotype by Df(3R)ry615. The j6E7 mutation was tested in the PP1 87B gene and dominant suppression of the png3318/png1058 phenotype was found with 78% multinucleated embryos compared to 41% in sibling controls. PP1 87B also suppresses females homozygous for the strong allele, png1058, to produce multinucleated embryos. This suppression could be direct in that PP1 could be responsible for dephosphorylating the substrates of the Png kinase. It is possible, however, given the pleiotrophic nature of PP1 87B phenotypes that it is suppressing indirectly. A lethal P-element insertion (l(2)k09822) in another serine/threonine protein phosphatase gene, PP2A 28D, the protein phosphatase 2A catalytic subunit gene at 28D, also dominantly suppress the png3318/png1058 phenotype with 89% multinucleated embryos compared to 31% in sibling controls. A similar degree of suppression was observed for the l(2)s5286 allele of PP2A 28D, and both alleles suppresses the strong png1058 phenotype as well. Reducing the activity of a phosphatase might generally increase mitotic activities in the mutant embryos, thereby suppressing the png phenotype (Lee, 2001).
The suppression of png, plu, and gnu by overexpressing cyclin B is not complete because ultimately nuclear divisions fail, and the nuclei continue to replicate and become polyploid. It is possible that Cyclin B is the sole target of the Png/Plu Complex and Gnu and that the levels of increased Cyclin B protein in the png, plu, and gnu mutants (via increased copies of the cyclin B gene) are not adequate for completion of all the S-M cycles. However, it seems more likely that, although Cyclin B is a key target, other targets of the Png/Plu complex and Gnu are also important. Cyclin A is a particularly good candidate for two reasons. Cyclin A protein levels are decreased in png, plu, and gnu mutants; for png, the decrease is in proportion to the strength of the allele. Decreasing the dosage of maternal cyclin A to one copy causes an increase in cycle time during the early embryonic divisions. In contrast, decreasing the dosage of cyclin B does not affect the timing of nuclear cycles, whereas it does affect microtubule dynamics. These observations led to the conclusion that Cyclin B controls cytoskeletal events during the S-M cycles, but Cyclin A controls the nuclear cycles. If this model is correct, Cyclin A may be a critical target for the influence of Png, Plu, and Gnu on the nuclear cycles (Lee, 2001).
No enhancement of the png phenotype by mutations in cyclin A was observed, and overexpression of cyclin A unexpectedly enhanced the phenotype. This latter result likely reflects the ability of excess Cyclin A to promote DNA replication, but it is not clear why a reduction in Cyclin A does not affect the png phenotype. Further delineation of the role of Cyclin A levels will likely emerge from identification of Png kinase substrates and elucidation of the mechanism by which Png influences Cyclin A and B protein levels (Lee, 2001).
There are several mechanisms by which Png, Plu, and Gnu could affect Cyclin A and B protein levels, including maternal transcription, mRNA stability or processing, translation, and cyclin protein stability. Mutations in the pathway that target mitotic cyclin proteins for destruction were examined. No suppression of the png mutant phenotype was observed by reducing the dosage of the two known activators of cyclin destruction, fzy or fzr. Similarly, mutation of an APC/C subunit or several genes affecting the ubiquitin pathway did not alter the png mutant phenotype. These negative results do not exclude a role for Png in controlling APC/C-mediated protein degradation, since the dosage reductions may not have reduced protein activity below a crucial threshold. Additional experiments will be required to evaluate how PNG affects Cyclin A and B protein levels (Lee, 2001).
Given the well-established role for Cyclin E in progression through S phase, it was surprising to find that decreasing the cyclin E gene dosage enhances the png phenotype. Consistent with this observation, a mutation of dacapo, a CDK2/Cyclin E kinase inhibitor, slightly suppresses png. In addition to promotion of S phase, other cell cycle roles have been ascribed to Cyclin E that might account for the enhancement of png by decreased cyclin E. For example, in Xenopus egg extracts, Cdk2/Cyclin E has been implicated in activation of Cdc2/Cyclin B for progression into mitosis. In Drosophila, ectopic expression of cyclin E induces post-transcriptional accumulation of mitotic cyclins. Whether decreasing the cyclin E gene dosage enhances png by causing a further decrease in Cyclin B was tested, but no additional decrease in Cyclin B protein levels in embryos from png3318/png1058; l(2)05206/+ females was detected by immunoblotting (Lee, 2001).
There are two precedents for how Cyclin E could affect DNA replication in a manner that would enhance png. A paradoxical role for Cdk2/Cyclin E as a negative regulator of DNA replication in Xenopus extracts has been described. In Drosophila, Cyclin E also may negatively regulate DNA replication, because hypomorphic cyclin E mutations cause endocycling nurse cells to undergo late DNA replication with accumulation of increased amounts of heterochromatic DNA. Thus decreased cyclin E could permit more unregulated DNA replication in png mutant embryos, possibly further impeding entry into mitosis and resulting in enhancement of the png phenotype (Lee, 2001).
Mutation of eIF-5A strongly suppresses png mutants. At present it is not possible to distinguish whether this is so because eIF-5A directly influences the same process as png ( i.e., Cyclin B protein levels) or whether the suppression results from compensatory changes in the cell cycle. Although no increase in Cyclin B protein was detected in png1058/png1058; eIF-5A/+ strains by immunoblotting, it is possible that Cyclin B increases sufficiently to account for the suppression seen or that its local concentration in the vicinity of the zygotic nuclei is increased. The difficulty in defining the relationship between eIF-5A and png is in large part due to the fact that the biological function of eIF-5A is not understood, and it is not clear that its primary function is in translation initiation (Lee, 2001).
At the transition from meiosis to cleavage mitoses, Drosophila requires the cell cycle regulators encoded by the genes giant nuclei (gnu), plutonium (plu) and pan gu (png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. The gnu gene encodes a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu; in other words, gnu functions upstream of pan gu and plu (Renault, 2003).
Rabbit anti-Gnu antiserum Rb86, raised against a synthetic peptide comprising aa117-131 is specific for Gnu and for Gnu-GFP but does not recognise a truncated product of the gnu mutant. The expression profile of a functional Gnu-GFP fusion protein under the control of the gnu promoter was examined by immunoblotting and detection with a monoclonal antibody against GFP. Gnu-GFP is expressed in ovaries and 0- to 3-hour embryos and in unfertilized eggs, but not in larval tissues or in adult testes. The epitope-tagged protein has very similar expression to native Gnu detected with an anti-peptide antiserum, but has a somewhat longer half-life in cleavage embryos. No Gnu was detected in embryos more than 1 hour after egg deposition, in larvae or in adult testes. The mobility of Gnu and of Gnu-GFP from ovaries is slower than from unfertilized eggs or embryos suggesting Gnu is post-translationally modified. The mobility of the embryonic isoform matches the predicted size of the fusion protein (54 kDa). GFP mobility in extracts from ovaries and embryos from a ubiquitin-driven GFP line were identical, therefore it is only the Gnu moiety of Gnu-GFP that is modified (Renault, 2003).
In ovary extracts with phosphatase inhibitors, the slow moving form of Gnu-GFP was observed. If phosphatase inhibitors are omitted from the ovary extraction, the amount of slow moving form is reduced in favour of the fast moving form with the same mobility as Gnu-GFP from embryos. To ascertain which protein phosphatases (PPs) are involved, specific inhibitors of serine/threonine protein phosphatases were tested for their ability to stabilize the slow moving form. Okadaic acid (OA) at low concentration (1 nM), sufficient to inhibit PP2A, does not stabilize slow moving Gnu-GFP, however OA at a higher concentration (50 nM), sufficient to inhibit PP1, stabilizes slow moving Gnu-GFP. I-2Dm, a specific inhibitor of PP1, also stabilizes slow moving Gnu-GFP. It is concluded that PP1 can dephosphorylate Gnu in ovary extracts (Renault, 2003).
To determine the developmental time-point at which Gnu dephosphorylation occurs, the genomic gnu-GFP transgene was crossed into mutant backgrounds that cause the oocyte to arrest development during meiosis [cortex and grauzone], or immediately following meiosis but prior to the first zygotic mitosis [deadhead]. Gnu-GFP mobility in ovaries and eggs in these mutant backgrounds was indistinguishable from wild-type indicating that Gnu is dephosphorylated before meiotic arrest induced by cortex and grauzone, most likely at egg activation (Renault, 2003).
In ovaries, Gnu-GFP is first observed in fixed oocytes of stage 11 egg chambers. In subsequent stages it accumulates in the oocyte but is not observed in nurse cells. In eggs, Gnu-GFP is cytoplasmic and shows no association with the replicatively inactive polar body chromosomes. In syncytial embryos Gnu-GFP is again cytoplasmic at all stages of the cell cycle. Although the nuclear envelope stains somewhat more distinctly, Gnu is neither strongly localized within, nor excluded from zygotic nuclei (Renault, 2003).
Gnu-GFP mobility in ovaries and eggs in both null and weak png backgrounds is indistinguishable from wild type. Therefore, Pan gu is neither the kinase that phosphorylates Gnu nor part of a pathway leading to Gnu dephosphorylation. Even if Pan gu does not influence Gnu modification, it might regulate Gnu localisation. To test this possibility Gnu-GFP localisation was examined in a png background. Gnu was found to be cytoplasmic in png embryos and it is excluded from the giant nuclei (Renault, 2003).
Gnu-GFP was mis-expressed in Drosophila ovaries using the UAS-GAL4 system. Females containing the maternal alpha4tubulin>GAL4:VP16 driver and UASp gnu-GFP are sterile and do not lay eggs. Staining of their ovaries reveals the expression of Gnu-GFP from stage 5 onwards. Egg chambers up to stages 8-10 have wild-type morphology. However subsequent stages are characterized by large amounts of irregularly localized, often fragmented, chromatin resulting from the degeneration of nurse cell nuclei. No stage 14 egg chambers could be distinguished. Suprisingly, given that Gnu-GFP, expressed from its own promoter, is unlocalized in embryos, mis-expressed Gnu-GFP is exclusively nuclear in nurse cells (Renault, 2003).
To test whether the sterility associated with Gnu mis-expression is a consequence of Gnu alone, Gnu was mis-expressed in ovaries in a png mutant background. Females homozygous for png1058 and containing the maternal alpha4tubulin>GAL4:VP16 and UASp gnu-GFP constructs lay eggs. Staining of their ovaries revealed they were morphologically normal with no abnormal egg chambers or fragmented DNA. The egg laying rates for such females are similar to wild type indicating that the restoration of ovarian function is complete. The eggs do not hatch but, when stained for chromatin; they exhibit a giant nuclei phenotype, typical of png embryos. The earliest mis-expression and amount of Gnu-GFP fluorescence in a png background is the same as in a wild-type background indicating that the restoration of ovary function is not caused by an effect of png on Gnu-GFP mis-expression levels or timing. The effect of a homozygous null plu mutation in combination with Gnu-GFP mis-expression is indistinguishable from that of the null png mutation (Renault, 2003).
Since the Gnu gain-of-function phenotype resembles that of Profilin mutants the actin cytoskeleton of egg chambers was examined. Defects were first seen at stage 10, when nurse cells mis-expressing Gnu fail to assemble the actin meshwork and do not dump their cytoplasmic contents into the oocyte. It is not know whether the disruption of actin reorganization is the primary consequence of excess Gnu, or whether premature egg chamber death results in the dramatically abnormal aggregates of F-actin. What is clear from these epistasis experiments is that this 'dump-less' phenotype is specific, in that it also requires Pan gu and Plu (Renault, 2003).
Thus, Gnu mis-expression using the UAS-Gal4 system in Drosophila ovaries results in sterility due to an inability to lay eggs. Dissection of the ovaries revealed that early oogenesis is unaffected. Gnu was expressed in egg chambers from stage 5 onwards and was localized solely to the nurse cell nuclei. No normal egg chambers could be discerned at stage 10 or later when gross aberrations in the organization of the actin cytoskeleton resulted in failure to transfer nurse cell cytoplasm into the oocyte. Gnu mobility from such ovaries is identical to the dephosphorylated form suggesting that the protein kinase that phosphorylates Gnu is not present or active in the nurse cells (Renault, 2003).
Since gnu, plu and png mutations have the same phenotype, it was previously not possible to determine whether the gene products act in series or in parallel. The dominant ovarian phenotype resulting from Gnu mis-expression was used to investigate the epistasis of gnu, png and plu. Loss of png or plu function blocked the ovarian phenotype caused by Gnu mis-expression. This result implies that ectopic Gnu destroys egg chambers only through Pan gu and Plu. It is therefore likely that wild-type Gnu function in the egg and embryo also requires Pan gu. Although Gnu-GFP is more obviously excluded from the larger png giant nuclei than from zygotic nuclei, the explanation that Gnu requires Pan gu for nuclear localization is not favored for at least two reasons. (1) Gnu-GFP is not specifically nuclear in wild-type embryos and (2) in a png null ovary, ectopic Gnu-GFP is able to concentrate in the polyploid nurse cell nuclei. The remaining possibilities are that Gnu acts upstream of Pan gu and Plu or that it acts in a complex with Pan gu and Plu (Renault, 2003).
Gnu was mis-expressed in polytene salivary glands and ovarian follicle cells. In both cases Gnu was exclusively nuclear, but its expression had no obvious effect on tissue morphology. However, not all nurse cell nuclei in an egg chamber contained Gnu-GFP, suggesting that the presence of Gnu-GFP reflects the transcriptional activity of the nucleus or depends upon its cell cycle status. Why is Gnu nuclear in polytene cells and cytoplasmic in the diploid syncytial blastoderm and larval neuroblasts? Gnu contains no obvious nuclear import sequence, suggesting that Gnu binds a factor that is cytoplasmic in eggs and embryos (including those with giant nuclei), and is nuclear in polytene tissues. Polytene and diploid tissues have different Cyclin profiles. Embryos are replete with maternal Cyclins A, B and E whereas polytene tissues have no Cyclin A or B and Cyclin E is expressed periodically in nurse cell nuclei and constantly in the germinal vesicle. These observations fit the Cyclin E pattern, ectopic Gnu is not present in all nurse or follicle cell nuclei and is concentrated in germinal vesicles (Renault, 2003).
The DNA replication in gnu embryos resembles the endoreduplication observed in Drosophila ovarian nurse cells and larval salivary glands and this raises the question of how the normal mechanisms that license DNA replication once per cell cycle are subverted in gnu embryos. In yeast, Cdk1 activity, modulated by Cyclin levels, is responsible for resetting replication origins raising the possibility that over-replication in gnu embryos may result from inappropriate Cdk1 activity. Indeed, in gnu, plu or png embryos, levels of Cyclin A and B proteins and Cdk1 activity are reduced. Restored Cyclin B levels can suppress a weak png mutation (Renault, 2003).
Several features of early Drosophila embryogenesis may have necessitated the evolution of these specialized regulators of the cell cycle: (1) distinct cell cycle fates befall the polar body and zygotic nuclei within a common cytoplasm; (2) many cell cycle regulators are in excess, so that the first 13 cycles are rapid and lack G1 or G2 phases, but S and M phases must alternate accurately and (3) correct cell cycle regulation is achieved by a small subset of the available cell cycle control proteins (e.g., Cyclins A and B). In this context, Gnu, Plu and Pan gu act coordinately to ensure that the cell cycle oscillations experienced by the nuclei are temporally and locally apt (Renault, 2003).
In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. Egg activation is necessary and sufficient to trigger transcript destabilization. Thirteen maternal-effect lethal loci have been identified that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. It is proposed that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation (Tadros, 2003).
A strong correlation exists between the S-to-M transition at the end of meiosis and the trigger for transcript instability: in vitro-activated eggs proceed normally through vitelline membrane reorganization, mRNA translation, and meiosis but then begin to show abnormalities at the S-to-M transition that follows meiosis. Unexpectedly, transcript destabilization fails to be triggered in these eggs. The simplest interpretation of this result is that transcript destabilization is triggered coincidently with this S-to-M transition and that it fails in in vitro-activated eggs because the temporal progression of egg activation is disrupted at this point. The fact that the least-pleiotropic RNA destabilization mutants (png, plu, and gnu) also proceed normally to this point and then fail to undergo both the S-to-M transition and transcript degradation is fully consistent with this interpretation (Tadros, 2003).
Previous analyses indicated that the S-to-M transition defect in png, plu, and gnu was likely to be a result of reduced cyclin-B levels and thus of reduced CDK activity. Several lines of evidence are presented that argue against a simple interpretation of the png, plu, and gnu mutant effects on transcript instability, namely, that the S-to-M transition and high CDK activity per se is required for transcript destabilization. Briefly, genetic suppression of the S-to-M transition defect in these mutants, under conditions in which CDK/cyclin activity is restored, does not result in suppression of the transcript degradation defect. Reciprocally, a severe reduction of CDK activity does not abrogate transcript destabilization. In addition, mutations that suppress the overreplication phenotype of the S-to-M transition mutants by means other than restoration of CDK/cyclin activity [e.g., by preventing replication as in fs(1)Ya mutants] do not suppress the transcript destabilization defect. These analyses have been extended by analyzing three additional dosage-sensitive suppressors of png: 1) eIF-5A, which is involved in translation initiation and replication and mutation of which, like that of fs(1)Ya, does not result in restoration of cyclin-B levels, and (2 and 3) two protein phosphatases, PpI-87B and Pp2A-28D. In all three cases, while the mitotic defect of png is clearly suppressed, the RNA degradation phenotype is indistinguishable from that of png single mutants (Tadros, 2003).
Png is a S/T kinase that is likely to be in a complex with Plu and Gnu. A model consistent with all of the above data is that transcript destabilization is triggered by the Png-PLu-Gnu signaling complex coincident with, but independent of, the complex's role in regulating the S-to-M transition at the end of meiosis. To test this hypothesis it will be necessary to carry out genetic screens for suppressors of the png, plu, or gnu transcript degradation phenotype per se, rather than relying upon suppressors of the mitotic phenotype as has been done in this study. Identification of molecular targets of the Png-Plu-Gnu signaling complex may lead to the definition of independent pathways through which transcript destabilization and the S-to-M transition are regulated (Tadros, 2003).
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date revised: 10 August 2018
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