Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, we identified two additional developmental amplicons in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression.

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein.Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.

In metazoan oocytes, a metaphase arrest coordinates the completion of meiosis with fertilization. Vertebrate mos maintains the metaphase II arrest of mature oocytes and prevents DNA replication between the meiotic divisions. We identified a Drosophila homolog of mos and showed it to be the mos ortholog by two additional criteria. The dmos transcripts are present in Drosophila oocytes but not embryos, and injection of dmos into Xenopus embryos blocks mitosis and elevates active MAPK levels. In Drosophila, MAPK is activated in oocytes, consistent with a role in meiosis. We generated deletions of dmos and found that, as in vertebrates, dmos is responsible for the majority of MAPK activation. Unexpectedly, the oocytes that do mature complete meiosis normally and produce fertilized embryos that develop, although there is a reduction in female fertility and loss of some oocytes by apoptosis. Therefore, Drosophila contains a mos ortholog that activates a MAPK cascade during oogenesis and is nonessential for meiosis. This could be because there are redundant pathways regulating meiosis, because residual, low levels of active MAPK are sufficient, or because active MAPK is dispensable for meiosis in Drosophila. These results highlight the complexity of meiotic regulation that evolved to ensure accurate control over the reproductive process.

Following completion of meiosis, DNA replication must be repressed until fertilization. In Drosophila, this replication block requires the products of the pan gu (png), plutonium (plu) and giant nuclei (gnu) genes. These genes also ensure that S phase oscillates with mitosis in the early division cycles of the embryo. We have identified the png gene and shown that it encodes a Ser/Thr protein kinase expressed only in ovaries and early embryos, and that the predicted extent of kinase activity in png mutants inversely correlates with the severity of the mutant phenotypes. The PLU and PNG proteins form a complex that has PNG-dependent kinase activity, and this activity is necessary for normal levels of mitotic cyclins. Our results reveal a novel protein kinase complex that controls S phase at the onset of development apparently by stabilizing mitotic cyclins.