Basic aspects of ovarian development in Drosophila melanogaster

Modern views of the development and structural organization of the female reproductive system in Drosophila melanogaster are reviewed. Special emphasis is placed on the generation and development of follicles in the germarium and the interactions of germline and somatic cells in the egg chamber. Detailed consideration is given to the main events that ensure and regulate the transport of mRNA, proteins, and organelles from nurse cells to the oocyte in the germarium and at later stages of egg chamber development.     

A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging

This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes approximately 24 h while the preparation of egg chambers for live imaging requires only 15-20 min.     

Complementation between alleles at the ovarian tumor locus of Drosophila melanogaster

The (ovarian tumor) otu gene resides at 23.2 on the genetic map of the X chromosome and near 7F1 on the cytological map. This germ line-expressed locus behaves as if it encodes a gene product which is required during certain steps in the transformation of oogonia into functional oocytes. On the basis of their ovarian morphologies 17 ethyl methane sulfonate (EMS)-induced mutants have been distributed among three developmental classes as follows: quiescent (eight), oncogenic (four), and differentiated (five). The otu13 and otu14 alleles interact to yield fertile females, and many other heteroallelic combinations show partial complementation. Since many mutant alleles interact beneficially, the functional product of the otu gene may be a multimer. We conclude, from an analysis of heteroallelic interactions and dosage effects, that the abnormal phenotypes observed are graded consequences of reduced levels of functional gene product and that the minimum concentration required for development increases as oogenesis proceeds.     

A gene necessary for late sperm function in Drosophila melanogaster.

A male-sterile mutant, ms(1)7, of Drosophila melanogaster is defective in post-ejaculatory sperm function. The mutant gene is located at one of at least five male fertility loci in 18F through section 20 of the polytene X-chromosome. It is proposed that the ms(1)7+ gene product modifies a component of the sperm head. A nearby gene may be functionally related to ms(1)7+.     

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

Na,K-ATPase and V-ATPase in ovarian follicles of Drosophila melanogaster.

Uncovering the cause and meaning of bioelectric phenomena in developing systems requires investigations of the distribution and activity of ion-transport mechanisms. In order to identify and localize ion pumps in ovarian follicles of Drosophila, we used immunofluorescence microscopy, immunoelectron microscopy, subcellular fractionation, immunoblots, and acridine-orange staining. We applied various antibodies directed against the Na,K-pump (Na,K-ATPase) and against vacuolar-type proton pumps (V-ATPase). During all phases of oogenesis, Na,K-ATPase were found in apical and lateral follicle-cell membranes and, during rapid follicle growth (beginning with stage 10), also in nurse-cell membranes and in the oolemma. V-ATPase were detected in various cytoplasmic vesicles and in yolk spheres and, beginning with stage 10, also in apical follicle-cell membranes and in the oolemma. Given these and earlier results, we propose that: 1) V-ATPase coupled to secondary active antiporters represent the ouabain-intensitive potassium pumps described previously; 2) both Na,K-ATPase and V-ATPase are involved in bioelectric phenomena as well as in osmoregulation and follicle growth, especially during stages 10-12; 3) organelle-associated V-ATPase play a role in vesicle acidification and in yolk processing; and 4) the channel-forming protein ductin is a component of both V-ATPase and gap junctions in ovarian follicles of Drosophila.     

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Apoptosis-mediated cell death within the ovarian polar cell lineage of Drosophila melanogaster

Polar cells have been described as pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. We show that definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, we demonstrate that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers.     

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

 In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process. Received: 12 December 1997 / Accepted: 6 January 1998     

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.     

Circadian clock properties of fruit flies Drosophila melanogaster exhibiting early and late emergence chronotypes

The role of circadian clocks in timing daily behaviors is widely acknowledged, and while empirical evidence suggests that clock period is correlated with the preferred phase of a rhythmic behavior (chronotype), other clock properties have also been hypothesized to underlie chronotype variation. Here, we report that fruit fly Drosophila melanogaster populations exhibiting evening emergence chronotype (late) are characterized by higher incidence of behavioral arrhythmicity in constant dim light, wider range of entrainment, reduced rates of re-entrainment to simulated jet-lag and higher amplitude of both entrained and free-running rhythms as compared to those exhibiting morning emergence chronotype (early). Our results thus highlight the role of circadian clock properties such as zeitgeber sensitivity, amplitude and coupling in driving chronotype variation.     

Alteration of chromosome structure in ovarian nurse cells of Drosophila melanogaster by hybrid dysgenesis

The impact of hybrid dysgenesis on the chromosome structure of Drosophila melanogaster ovarian nurse cells was studied. In the examined lines and interlinear hybrids (including those yielded by dysgenic crosses in the P-M and I-R systems of hybrid dysgenesis), disturbed chromosome synapsis was revealed. The disturbance was somewhat similar to that observed in interspecific hybrids. Quantitative analysis showed that the mean frequency of nuclei with defective chromosome pairing ranged from 60.4 to 76%. FISH analysis of ovarian nurse chromosomes of Canton S x Berlin hybrids showed differences in the label localization in asynaptic homologs of arm 2L, which probably results in disrupted homolog pairing and reveal interlinear differences in localization of mobile genetic elements. Our results conform to Sved's model stating that hybrid dysgenesis is based on disorganization of the germline nuclear space.     

Identification and genetic localization of mRNAs from ovarian follicle cells of Drosophila melanogaster.

RNA synthesis in ovarian follicles of Drosophila melanogaster was studied by methods which eliminate experimentally induced alterations in gene expression. Gel electrophoresis of follicular RNA, labeled after injection of precursors into females, revealed qualitative and quantitative differences in synthesis during the course of oogenesis. A highly heterogeneous group of poly(A)-containing RNAs is produced during much of the course of follicular development. However, post-vitellogenic stages synthesize a small number of stage-specific poly(A)-containing RNAs. During this period, RNA synthesis is known to take place primarily in the follicle cells, which are engaged in the production of the endochorion and exochorion. Two intense bands of nonmitochondrial poly(A)⁺ RNA are labeled between stage 11 and early stage 13. The synthesis of a more heterogeneous group of very small poly(A)-containing RNAs characterizes the last part of oogenesis, stages 13 and 14. Evidence is presented to show that these RNAs are specifically localized in the follicle cells of the egg chamber. We propose that they represent mRNAs for chorion proteins. In situ hybridization of preparations of late stage poly(A)-containing RNA to salivary gland chromosomes revealed two major sites of complementarity, 7E11 and 12E, as well as several minor sites. Experiments in which RNAs were separated on gels prior to hybridization in situ suggested that both the major stage 12-specific RNA bands contained molecules which were complementary to DNA in the 7E11 region. It is particularly interesting that this site is within a small chromosomal interval known to contain the gene ocelliless. Females homozygous for ocelliless have been shown to produce structurally abnormal chorions (Johnson and King, 1974).     

Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster

Summary An ultrastructural study was made of the ring canal system which connects the sister ovarian cystocytes that arise in the germaria of wild type Drosophila melanogaster females. It was discovered that during an oogonial mitosis both chromosomes and spindle are enclosed by a multilayered, perforated membrane system derived (at least in part) from the nuclear envelope. The cytokinesis of stem line oogonia takes place through the formation of a cleavage furrow. A second method of formation of plasma membrane is found in the case of cystocytes. It involves the production along the plane of division of a plaque of interconnected vesicles and tubules and later the coalescence of nearby tubules to form continuous sheets of membrane which segregate the cytoplasms of the sister cells. However, these remain connected by a canal which is enclosed by a ring-shaped rim that is completed prior to the plasma membrane to which the rim is subsequently attached. It is postulated that the rim represents a transformed midbody. As development proceeds the canal becomes wider, its rim becomes thicker, and the inner circumference of the rim becomes coated with a thick deposit having different cytochemical properties than the rim itself. Cystocyte divisions produce sister cells which differ in that one receives all previously formed canals; the other none. In the case of the last division (and perhaps in earlier ones as well) the sister cell receiving all previously formed canals also receives more cytoplasm than its sister. As the cells of the cluster grow, the canals remain close together. This finding suggests that when new plasma membrane is synthesized, it is added in areas remote from the canals. An investigation of the positioning in three dimensions of the fifteen canals of a newly formed, 16 cellcluster suggests that the spindles produced at one division are never parallel to those formed at the subsequent division. This continual shifting of the axes of the spindles at consecutive divisions presumably results in the branching chains of cells which characterize a cystocyte cluster. The possession of a unique pattern of cortical structures by two cystocytes is accompanied by the nuclear synthesis of synaptonemal complexes. The other fourteen cystocytes differentiate into nurse cells. In the most posterior portion of the germarium one of the two potential oocytes switches to the nurse cell developmental pathway. This switched off oocyte and the definitive oocyte grow at rates which differ greatly and are correlated to the amount of contact between their surfaces and certain follicle cells. As development proceeds centrioles accumulate in the oocyte, and most of these are thought to have been carried from the nurse cells into the oocyte in the nutrient stream.The authors are grateful to Richard Z. Belch and James E. Bradof for their conscientious assistance and to E. John Pfiffner for preparation of the inked drawings and construction of the Polyform models. This research was supported by the National Science Foundation grant GB7457.     

Mass fractionation of Drosophila egg chambers.

Egg chambers from Drosophila ovaries may now be fractionated in large quantities suitable for biochemical work. A suspension of isolated egg chambers is prepared by subjecting ovaries to digestion with 0.067% collagenase. Egg chambers are separated in size classes (which correspond to different developmental stages) by sieving them through a column of superimposed nylon nets. The method can, in principle, be used for the separation of any particles ranging upward from 10 μm in diameter.     

Meiosis in Drosophila melanogaster

Chromosome pairing during meiosis I in D. melanogaster males was investigated ultrastructurally by examining complete bivalents in electron micrographs of serial thin sections. The XY bivalent is characterized by the presence of unique material located between the two half-bivalents at the site of synapsis. The material has a fibrillar appearance and is less electron dense than the surrounding chromatin. YY bivalents in XYY males and XY bivalents containing the X chromosome, In(1)sc^4Lsc^8R, where the pairing sites of the X chromosome are inverted and partially deleted also possess this material. The material is not associated with autosomal bivalents and may represent a morphological manifestation of the hypothetical cohesive elements (collochores) which are thought to function in conjunction of the X and Y chromosomes (Cooper, 1964).