DNA repair in Drosophila oogenesis

In experiments with females of lines with an impaired DNA repair systems mei-9 (impaired excision repair) and mei-41 (impaired postreplicative repair), a method of successive irradiation by X-rays (1000 R) and hyperthermia (+37 degrees C) action was used for the purpose of defining a moment when DNA repair takes place in oogenesis. Repair in mature mei-41 oocytes judged of by synergism effect of the both factors acting was ascertained to take place right after X-raying (prior to DNA replication) and being absent at the fertilization period (at the time of or after DNA replication). DNA repair in mei-9 females was not registered in both cases. On the basis of these facts, it is suggested that coordination of various DNA repair systems is necessary for damaged chromosomes to be repaired. It is also concluded that the method used can be regarded as an effective technique in the study of mutation process.     

Ultrastructural observations on oogenesis in Drosophila

The ultrastructure of the follicle cells and oocyte periplasm is described during the stages of oogenesis immediately prior to, during, and immediately subsequent to, vitellogenesis. A number of features have not been described previously in Drosophila. Some yolk appears prior to pinocytosis of blood proteins. However, most of the protein yolk forms while the periplasm is filled with micropinocytotic invaginations and tubules derived from the oolemma. These tubules retain the internal layer of material characteristic of coated vesicles and are found to fuse with yolk spheres. No accumulation of electron-dense material in the endoplasmic reticulum or Golgi of the oocyte is found. Both trypan blue and ferritin are accumulated by the oocyte. The follicle cells have an elaborate endoplasmic reticulum during the period of maximum yolk accumulation. Adjacent cells are joined at their base by a zonula adhaerens, forming a band around the cells, and by plaques of gap junctions. Gap junctions are also present between nurse cells and follicle cells. During chorion formation, septate junctions also appear between follicle cells, adjacent to the zonula adhaerens.     

Stage-specific apoptotic patterns during Drosophila oogenesis

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.     

Drosophila oogenesis: versatile spn doctors

Recent work on Drosophila oogenesis has uncovered connections between cell-cycle checkpoints and pattern formation. Genes of the spindle class, which encode double-strand break repair enzymes and RNA helicases, affect oocyte polarity and the decision whether to differentiate as an oocyte or a nurse cell.     

Monitoring positional information during oogenesis in adult Drosophila

About 184P[lac, ry+]A insertions (O'Kane & Gehring, 1987) have been incorporated into the genome via P element-mediated transformation. The temporal-spatial localization of beta-galactosidase, synthesized by these insertions during oogenesis, is described. 32% present control levels of endogenous beta-galactosidase expression and 68% show novel patterns. 13% of the insertions are germline-specific; 33%, follicle-cell-specific; 20% are expressed in both germ line and follicle cells; and 2%, specific to the germarium. Several lines exhibit strict temporal-spatial localizations of beta-galactosidase; notably those expressed in specific populations of follicle cells. The results are discussed with respect to some of the positional information encoded in the genome to which the insertions respond, the use of the insertions as markers for cell differentiation and the potential of the technique for isolating new genes involved in egg production.     

Quantifying the Gurken morphogen gradient in Drosophila oogenesis

Quantitative information about the distribution of morphogens is crucial for understanding their effects on cell-fate determination, yet it is difficult to obtain through direct measurements. We have developed a parameter estimation approach for quantifying the spatial distribution of Gurken, a TGFalpha-like EGFR ligand that acts as a morphogen in Drosophila oogenesis. Modeling of Gurken/EGFR system shows that the shape of the Gurken gradient is controlled by a single dimensionless parameter, the Thiele modulus, which reflects the relative importance of ligand diffusion and degradation. By combining the model with genetic alterations of EGFR levels, we have estimated the value of the Thiele modulus in the wild-type egg chamber. This provides a direct characterization of the shape of the Gurken gradient and demonstrates how parameter estimation techniques can be used to quantify morphogen gradients in development.     

Histone methylation is required for oogenesis in Drosophila

SET domain proteins are histone lysine methyltransferases (HMTs) that play essential roles in development. Here we show for the first time that histone methylation occurs in both the germ cells and somatic cells of the Drosophila ovary, and demonstrate in vivo that an HMT, the product of the eggless (egg) gene, is required for oogenesis. Egg is a SET domain protein that is similar to the human protein SETDB1 and its mouse ortholog ESET. These proteins are members of a small family of HMTs that contain bifurcated SET domains. Because depletion of SETDB1 in tissue culture cells is cell-lethal, and an ESET mutation causes very early periimplantation embryonic arrest, the role of SETDB1/ESET in development has proven difficult to address. We show that egg is required in the Drosophila ovary for trimethylation of histone H3 at its K9 residue. In females bearing an egg allele that deletes the SET domain, oogenesis arrests at early stages. This arrest is accompanied by reduced proliferation of somatic cells required for egg chamber formation, and by apoptosis in both germ and somatic cell populations. We propose that other closely related SET domain proteins may function similarly in gametogenesis in other species.     

Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis

Exit from the cell cycle requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit the division program after four mitotic divisions. We find that in encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the SCF pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. We show that Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of Cul1, phosphorylated Cyclin E and the proteasome 19S-RP subunit S1 with the fusome is affected in encore mutant germaria. We propose that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis.     

The role of the otu gene in Drosophila oogenesis

The ovarian tumor (otu) gene behaves as if it encodes a product (OGP) which is required during several early steps in the transformation of oogonia into functional oocytes. The ovarian phenotypes produced by various EMS-induced mutations can be explained as graded responses by individual mutant germ cells to the different levels of functionally active OGP they themselves synthesize. In addition, genetic evidence suggests that otu also encodes a second product that is utilized late in oogenesis. Molecular studies of the otugene demonstrate that it does transcribe at least two ovaryspecific RNAs. The possible function of the otu product in the cellular divisions that give rise to the oocyte and its sister nurse cells is discussed.     

bullwinkle and shark regulate dorsal-appendage morphogenesis in Drosophila oogenesis

bullwinkle (bwk) regulates embryonic anteroposterior patterning and, through a novel germline-to-soma signal, morphogenesis of the eggshell dorsal appendages. We screened for dominant modifiers of the bullwinkle mooseantler eggshell phenotype and identified shark, which encodes an SH2-domain, ankyrin-repeat tyrosine kinase. At the onset of dorsal-appendage formation, shark is expressed in a punctate pattern in the squamous stretch cells overlying the nurse cells. Confocal microscopy with cell-type-specific markers demonstrates that the stretch cells act as a substrate for the migrating dorsal-appendage-forming cells and extend cellular projections towards them. Mosaic analyses reveal that shark is required in follicle cells for cell migration and chorion deposition. Proper shark RNA expression in the stretch cells requires bwk activity, while restoration of shark expression in the stretch cells suppresses the bwk dorsal-appendage phenotype. These results suggest that shark plays an important downstream role in the bwk-signaling pathway. Candidate testing implicates Src42A in a similar role, suggesting conservation with a vertebrate signaling pathway involving non-receptor tyrosine kinases.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

Drosophila oogenesis: coordinating germ line and soma

A new function for Delta-Notch signaling has been discovered in Drosophila oogenesis: Delta expressed in the germ cells activates Notch in the surrounding somatic follicle cells to control their differentiation, proliferation and morphogenesis.     

Drosophila oogenesis: generating an axis of polarity

New studies of moesin function during Drosophila oogenesis show that Dmoesin tethers actin to the oocyte membrane. Interestingly, Dmoesin and an intact cortex are also required to stabilise ooycte polarity.     

Illuminating the role of caspases during Drosophila oogenesis

Cell death is a prominent feature of animal germline development. In Drosophila, the death of 15 nurse cells is linked to the development of each oocyte. In addition, females respond to poor environmental conditions by inducing egg chamber death prior to yolk uptake by the oocyte. To study these two forms of cell death, we analyzed caspase activity in the germline by expressing a transgene encoding a caspase cleavage site flanked by cyan fluorescent protein and yellow fluorescent protein. When expressed in ovaries undergoing starvation-induced apoptosis, this construct was an accurate reporter of caspase activity. However, dying nurse cells at the end of normal oogenesis showed no evidence of cytoplasmic caspase activity. Furthermore, although expression of the caspase inhibitors p35 or Drosophila inhibitor of apoptosis protein 1 blocked starvation-induced death, it did not affect normal nurse cell death or overall oogenesis in well-fed females. Our data suggest that caspases play no role in developmentally programmed nurse cell death.     

The Mirror transcription factor links signalling pathways in Drosophila oogenesis

Many genetic cascades are conserved in evolution, yet they trigger different responses and hence determine different cell fates at specific times and positions in development. At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and we show that this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (EGFR) signalling pathway to the Fringe signalling pathway via Mirror. The EGFR pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. We used twist as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to similar phenotypes to the ectopic expression of the activated form of EGFR. Thus, mirror also controls the Toll signalling pathway, leading to Dorsal nuclear transport. In summary, we show that the Mirror homeodomain protein provides a link that coordinates the Gurken/EGFR signalling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo.