Mutations in supernova,indicate that this gene is required for the division of germ line cells in Drosophila

Mutations in supernova, previously shown to uncouple chromosome replication from segregation during cleavage in Drosophila embryos, also sanctions extra divisions of cystoblasts and spermatoblasts. This leads either to the formation of egg chambers which contain more than fifteen nurse cells or testes which have an excess of spermatocytes. In maturing egg chambers two potential oocytes may be specified in which case they are often ectopically located and connected with surrounding nurse cells by four ring canals. However, a typical oocyte nucleus is not always present and these chambers usually become necrotic and degenerate. The nurse cells are of variable size, but are still interconnected by a system of ring canals. They all possess a polyploid nucleus. Sequestering of maternal mRNA's from the nurse cells into the potential oocyte(s) takes place but there is no localization of this maternal information within the oocyte probably because of defective microtubule assembly. Many spermatocytes fail to complete meiosis so that bundles of spermatids are reduced in size and the males have reduced fertility. It is proposed that this gene is indirectly involved in regulating the timing of mitotic divisions in both cystoblasts and spermatoblasts through its interference with microtubule assembly which is consistent with its role during embryogenesis.     

Eggs over easy: cell death in the Drosophila ovary

Programmed cell death is the most common fate of female germ cells in Drosophila and many animals. In Drosophila, oocytes form in individual egg chambers that are supported by germline nurse cells and surrounded by somatic follicle cells. As oogenesis proceeds, 15 nurse cells die for every oocyte that is produced. In addition to this developmentally regulated cell death, groups of germ cells or entire egg chambers may be induced to undergo apoptosis in response to starvation or other insults. Recent findings suggest that these different types of cell death involve distinct genetic pathways. This review focuses on progress towards elucidating the molecular mechanisms acting during programmed cell death in Drosophila oogenesis.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Importin-alpha 2 is critically required for the assembly of ring canals during Drosophila oogenesis

The interstitial deletion D14 affecting the importin-alpha 2 gene of Drosophila, or imp-alpha 2(D14), causes recessive female sterility characterized by a block of nurse cell-oocyte transport during oogenesis. In wild-type egg chambers, the Imp-alpha 2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-alpha 2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-alpha 2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-alpha 2(D14) ovaries reveals that the block of nurse cell-oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-alpha2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-alpha 2(D14) egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-alpha 2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly.     

A stereological analysis of developing egg chambers in the honeybee queen,Apis mellifera

Summary A polytrophic ovariole of the queen honeybee, Apis mellifera, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each of these components, as a function of the position of the egg chamber in the ovariole. An oocyte increases in volume from approximately 8.9 × 10³ m³ to approximately 9.6 × 10⁶ m³ over an average series of 20 egg chambers.     

Electrically mediated protein movement inDrosophila follicles

Summary Distribution of rhodamine-conjugated lysozyme injected into the sixteen-cell syncytium comprising the germ-line portion of theDrosophila follicle is shown to be affected by charge. Positive molecules are able to migrate through intercellular bridges from the oocyte to the nurse cells, but are unable to migrate detectably from nurse cells to the oocyte. Their negatively charged counterparts can move from the nurse cells to the oocyte, but are unable to traverse the intercellular bridges in the counter direction. This charge-dependent movement of molecules is accompanied by an electrical potential difference, focused across the nurse cell-oocyte bridges, which makes the nurse cells negatively charged to the oocyte. The addition of insect hemolymph to the physiological salt solution in which the experiments were performed resulted in only a small increase in the transmembrane resistance, but enhanced the potential difference between oocyte and nurse cells from 0.2±0.3 (SE) mV (nurse cells negative) to 2.3±0.45 (SE) mV (nurse cells negative). Supported by NSF Grant # DB-18617     

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.     

Follicle cell development is partly independent of germ-line cell differentiation in Drosophila oogenesis

Summary The developmental potential of the cells of the somatic follicular epithelium (follicle cells) was studied in mutants in which the differentiation of the germ-line cells is blocked at different stages of oogenesis. In two mutants, sn ^36a and kelch, nurse cell regression does not occur, yet the follicle cells around the small oocyte continue their normal developmental program and produce an egg shell with micropylar cone and often deformed operculum and respiratory appendages. Neither the influx of nurse cell cytoplasm into the oocyte nor the few follicle cells covering the nurse cells are apparently required for the formation of the egg shell. In the tumor mutant benign gonial cell neoplasm (bgcn) the follicle cells can also differentiate to some extent although the germ-line cells remain morphologically undifferentiated. Vitelline membrane material was synthesized by the follicle cells in some bgcn chambers and in rare cases a columnar epithelium, which resembled morphologically that of wild-type stage-9 follicles, formed around the follicle's posterior end. The normal polarity of the follicular epithelium that is characteristic for mid-vitellogenic stages may, therefore, be established in the absence of morphologically differentiating germ-line cells. However, the tumorous germ-line cells do not constitute a homogeneous cell population since in about 30% of the analyzed follicles a cell cluster at or near the posterior pole can be identified by virtue of its high number of concanavalin A binding sites. This molecular marker reveals an anteroposterior polarity of the tumorous chambers. In follicles mutant for both bgcn and the polarity gene dicephalic the cluster of concanavalin A-stained germ-line cells shifts to more anterior positions in the follicle.     

chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.

The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport. We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin. The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells.     

Effects of starvation on reproduction of the predacious mite <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae)

Effects of starvation on gravid females of Neoseiulus californicus were investigated at 20°C and 85% RH. When females that had been reared with abundant prey were swapped, just after laying their first egg, to conditions without any prey and water, they laid 1.8 eggs and survived for 4.3 days. In the body of well-fed females, an egg with eggshell and/or two oocytes were observed in the ventral and dorsal regions, respectively. The larger oocyte had two roundish nuclei and abundant yolk granules, and was enveloped with a vitelline membrane. These two nuclei were not fused but were just close to each other. The smaller oocyte had a nucleus, but had not yet formed yolk granules and vitelline membrane. Females after 12 h starvation had an egg in the ventral region and an oocyte in the dorsal region of the body. After more than 24 h starvation females maintained an oocyte in the dorsal region of the body, but had no egg in the ventral region. The oocyte was filled with abundant yolk granules and contained two irregular nuclei when females were starved for 24 h, but when starved for more than 36 h it contained one irregular nucleus. These findings suggest that (1) gravid females maintained an oocyte in the dorsal region after laying two eggs during starvation, (2) the oocyte was not absorbed during starvation, (3) the oocyte advanced vitellogenesis and the fusion of two nuclei, and (4) the vitellogenic oocyte was not enveloped with an eggshell and had not started embryogenesis.     

Molecular and genetic description of a new hypomorphic mutation of Trithorax-like gene and analysis of its effect on Drosophila melanogaster oogenesis

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional protein GAGA involved in many cellular processes. We have isolated and described a new hypomorphic mutation of The Trl gene—Trl _en82 . The mutation is the insertion of a 1.4 kb P-element into the 5′ untranslated region. Trl expression decreased in the ovaries of mutant flies by about 30%; however, it caused abnormalities. The Trl _en82 mutation combined with the null allele of Trl caused female sterility: the females laid a few small eggs with abnormal shape. Many egg chambers demonstrated abnormalities in the Trl _en82 mutants: the oocyte had a regular shape and intruded into the egg chamber region with nurse cells; the rapid transport of nurse cell cytoplasm into the oocyte was disturbed, which resulted in the “dumpless” phenotype of the chambers in mutants; follicular cells often did not completely cover the oocyte and concentrated on its posterior end; and the migration of centripetal cells was affected. We propose that the sterility of the Trl _en82 females is due to the abnormal functioning of follicular cells resulting from low Trlexpression. This proposal is confirmed by normalizing the mutant phenotype of Trl _en82 females after the transfection of Trl cDNA. Note that even an insignificant decrease in Trl expression in such females seriously affected the somatic cell functioning, while a significant decrease in its expression in strong hypomorphic mutants affected both somatic and germline cells in the egg chambers.     

Peculiarities of the organization of egg chambers in carabid ground beetles and their phylogenetic implications

The ovaries of 31 species of the coleopteran familyCarabidae have been studied by light and electron microscopy. Ovarioles of all the examined insects are of the polytrophic type. In the majority of the species a constant number of nurse cells per egg chamber has been observed. However, several species do not obey the 2(n) rule and the number of nurse cells varies considerably even in the consecutive egg chambers of the same ovariole. In spite of the differences, the number of intercellular bridges connecting nurse cells to the oocyte is fixed and species specific. InCarabidae seven types of egg chambers have been characterized regarding the number of divisions, the number of nurse cells and the way the nurse cells are bridged to the oocyte. Some phylogenetic implications are considered.     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

The role of juvenile hormone in the control of reproductive function in Drosophila virilis under nutritional stress

To investigate the role of juvenile hormone (JH) in the control of Drosophila reproduction under stress, JH degradation and reproduction were studied under nutritional stress and JH treatment in Drosophila virilis females of wild type (wt) and a heat stress (hs) mutant: this mutant does not respond to heat stress by alterations in JH metabolism and has decreased JH level and fertility under normal conditions. One day of starvation results in a decrease of JH degradation, a delay in oocyte maturation, degradation of early vitellogenic egg chambers, accumulation of mature oocytes and a 24 h oviposition arrest in both wt and hs females. A fertility decrease was observed in both wt and hs females 24 h following the end of starvation. JH treatment leads to a decrease of JH degradation and an arrest of oviposition for 24 h in fed females. JH treatment prior to starvation seems to protect some oocytes from resorption: in JH-treated wt females, fertility increases rapidly following the end of starvation. The dynamics of JH degradation and fertility are similar following starvation and JH treatment. The role of JH in the accumulation of mature oocytes and the delay of oviposition under stress are discussed.     

Observations on the polarity of mutant Drosophila follicles lacking the oocyte

Summary Homozygous females of the mutantsegalitarian andBicaudal-D ^R26produce follicles in which the oocyte is replaced by an additional nurse cell. Normal morphological markers for polarity can be identified in mutant follicles but the normal spatial organization of these markers is disturbed. For example, nurse-cell nuclei of different ploidy classes are present but, contrary to wild-type follicles, the nuclei show no anteroposterior ploidy gradient. The two cells with four intercellular bridges, one of which should have developed into the oocyte rather than a nurse cell, are located at the posterior pole only in young follicles (up to about stage 5), whereas during later stages they are more often found at lateral or intermediate positions. This disturbed polarity correlates with a variable aberrant pattern of extracellular ionic currents. Moreover, in the mutant follicles patches of columnar follicular epithelium differentiate locally although this type of epithelium forms normally only around the oocyte. The follicle cells at both follicle poles possess anterior quality since they migrate from both poles towards the centre of the follicle, as do the border cells restricted to the anterior pole in wild-type follicles. Our analysis indicates that in the mutants the follicular polarity is normal at first but becomes disturbed during stages 5 to 6. The secondary breakdown of polarity is likely to follow on from the absence of the oocyte.     

Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase

During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

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     

Immunologie localization of α- and β-endorphins and β-lipotropin in corticotropic cells of the normal and anencephalic fetal pituitaries

Summary Intercellular bridges have been detected in ovarian follicle cells of Drosophila melanogaster. These bridges occur widely between follicle cells of previtellogenic chambers, while, in vitellogenic chambers, they become restricted to the columnar follicle cells. Usually, only one bridge is detectable between adjacent follicle cells, but a single cell may form two cytoplasmic continuities.The fine structure of the intercellular bridges is similar to that previously described in the development of Drosophila. The bridge wall consists of two layers of which the more external is more electron dense and thinner than the inner one.The role played by the intercellular bridges in the determination of a synchronous differentiation of the linked follicle cells is discussed in relation to the known behaviour of these cells in the secretion of the egg covering precursors.