Johannes Bohrmann

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K+], high intracellular [Ca2+], octanol, dinitrophenol, and NaN3, but not with retinoic acid, basic pH, or high extracellular [Ca2+]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.

Drosophila unconventional myosin VI is involved in intra- and intercellular transport during oogenesis.

Abstract. During mid-oogenesis of Drosophila, cyto plasmic particles are transported within the nurse cells and through ring canals (cytoplasmic bridges) into the oocyte by means of a microfilament-dependent mecha nism. Video-intensified fluorescence timelapse mi croscopy, in combination with microinjections of antibodies directed against Drosophila 95F myosin, have revealed that this unconventional myosin of class VI is involved in the transport processes. The results indicate that certain cytoplasmic particles in the nurse cells move along microfilaments due to their direct association with myosin VI motors. Additional myosin- VI molecules located at the rim of the ring canals seem to be involved in particle transport into the oocyte. Microinjected mitochondria-specific dyes have revealed that some of these particles are mitochondria.

Gap junctions in the ovary of Drosophila melanogaster: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels

BackgroundIn the Drosophila ovary, germ-line and soma cells are interconnected via gap junctions. The main gap-junction proteins in invertebrates are members of the innexin family. In order to reveal the role that innexins play in cell-cell communication during oogenesis, we investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and analyzed follicle development following channel blockade.ResultsWe found innexin 1 predominantly localized to the baso-lateral domain of follicle cells, whereas innexin 2 is positioned apico-laterally as well as apically between follicle cells and germ-line cells. Innexin 3 was observed laterally in follicle cells and also in nurse cells, and innexin 4 was detected in the oolemma up to stage 8 and in nurse-cell membranes up to stage 12. In order to test whether innexins form channels suitable for intercellular communication, we microinjected innexin antibodies in combination with a fluorescent tracer into the oocyte of stage-10 follicles. We found that dye-coupling between oocyte and follicle cells was largely reduced by innexin-2 antibodies directed against the intracellular C-terminus as well as against the intracellular loop. Analyzing in vitro, between stages 10 and 14, the developmental capacities of follicles following microinjections of innexin-2 antibodies revealed defects in follicle-cell differentiation, nurse-cell regression, oocyte growth and choriogenesis.ConclusionOur results suggest that all analyzed innexins are involved in the formation of gap junctions in the ovary. While innexins 2 and 3 are colocalized between soma cells, innexins 2 and 4 are colocalized between soma and germ-line cells. Innexin 2 is participating in cell-cell communication via hemichannels residing in the oolemma. It is obvious that gap-junctional communication between germ-line and soma cells is essential for several processes during oogenesis.

Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles

BackgroundBioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis.ResultsTo obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca2+-channels, amiloride-sensitive Na+-channels and Na+,H+-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca2+-channels were found, suggesting a mechanism for transducing bioelectric signals into cellular responses. Moreover, gradients of electrical potential and pH were observed within single cells.ConclusionsOur data suggest that spatial patterning of Vmem, pHi and specific membrane-channel proteins results in bioelectric signals that are supposed to play important roles during oogenesis, e. g. by influencing spatial coordinates, regulating migration processes or modifying the cytoskeletal organization. Characteristic stage-specific changes of bioelectric activity in specialized cell types are correlated with various developmental processes.

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‐ATPases). During all phases of oogenesis, Na, K‐ATPases 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‐ATPases 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‐ATPases coupled to secondary active antiporters represent the ouabain‐insensitive potassium pumps described previously; 2) both Na, K‐ATPases and V‐ATPases are involved in bioelectric phenomena as well as in osmoregulation and follicle growth, especially during stages 10–12; 3) organelle‐associated V‐ATPases play a role in vesicle acidification and in yolk processing; and 4) the channel‐forming protein ductin is a component of both V‐ATPases and gap junctions in ovarian follicles of Drosophila.

In vitro culture ofDrosophila ovarian follicles: The influence of different media on development, RNA synthesis, protein synthesis and potassium uptake

SummaryContradictory electrophysiological results were recently obtained inDrosophila ovarian follicles kept in different salines or complete media during measurements. Therefore, I checked follicles maintained in various solutions using morphological, physiological and biochemical criteria. Defined complete media were the best for supporting development from stage 10 to stage 14 (end of oogenesis). Supplementation of the solutions with insect pupal haemolymph had negative effects. For the maintenance of RNA synthesis, complete media were again superior to simple salines. Total protein synthesis was not very sensitive to the culture conditions during short incubation periods, but electrophoretic protein patterns were slightly less complex in the salines than in complete media. Furthermore, some major proteins (e.g. chorion proteins) synthesized during longterm culture failed to appear in the salines. In view of extrafollicular electrical currents and intracellular electrical potentials, potassium uptake experiments were conducted with several inhibitors, using rubidium-86 as a probe. Both potassium concentration and osmolarity were found to exert strong influences on total potassium uptake of the follicles. In the tested media and salines differing amounts of potassium were taken up via (Na+, K+)-ATPase, via other K+-pump(s) or passively. The possible influences of several parameters on the outcome of earlier in vitro experiments withDrosophila follicles are discussed. For further in vitro studies the chemically defined complete R-14 medium seems to be the most suitable.

Inverse correlation between mean nuclear DNA content and cell number in nurse cell clusters of Drosophila

In the Drosophila ovarian follicle, 15 polyploid nurse cells (NC) are linked by cytoplasmic bridges to the anterior pole of the oocyte. The mutant dicephalic occasionally produces aberrant follicles whose 15 nurse cells are separated into two clusters, each attached to one oocyte pole [split nurse cell (SNC) follicles]. Feulgen cytophotometry of isolated nuclei from such clusters and from normal follicles of the same females revealed an inverse correlation between NC number per cluster and mean DNA content per nucleus. The maximum DNA content of nuclei from split NC clusters falls into the 4,096 C class as opposed to the 2,048 C class in 15 NC clusters; the mean DNA content per NC nucleus is 1,940 C in the former and 1,340 C in the latter. These correlations are confirmed by measurements on follicles containing fewer or more than 15 NC, and on SNC follicles from another mutant (spindel C). These findings indicate that replication in NC nuclei is controlled with reference to the transcriptional capacity of the individual NC cluster rather than the total capacity available per oocyte or follicle. We discuss this finding with respect to embryonic patterning as influenced by nurse cell location and list and discuss conflicting published reports on over- and underreplication in Drosophila follicles.

Potassium uptake into Drosophila ovarian follicles: Relevance to physiological and developmental processes

Abstract The origin of bioelectric phenomena in insect ovarian follicles is scarcely known and the presumption that these phenomena play an important role during development is still controversial. In the present study the K+ uptake mechanisms of vitellogenic Drosophila follicles were investigated in vitro using rubidium-86 as a probe. Follicles were exposed to the inhibitors 2,4-dinitrophenol, sodium azide, ouabain, 4-aminopyridine, tetraethylammonium chloride and barium chloride. The total K+ uptake changed stage-specifically and was different in anterior and posterior follicle halves. Maximal values were reached during mid-vitellogenesis (stage 10B), and at this stage K+ uptake into the posterior half (occupied by the oocyte with its columnar follicle cell epithelium) exceeded that into the anterior half (nurse cells with flat follicle cells) by 20%. This difference was probably due to ouabain-insensitive K+-pump(s) whereas the (Na+, K+)-pump was equally active in anterior and posterior halves and did not show stage-specific variations. The active K+ uptake into the anterior half increased during nurse-cell regression (stages 11 and 12). Active as well as passive K+ uptake increased with increasing extracellular pH. The used K+-channel blockers blocked K+ efflux but affected the passive K+ uptake only slightly. A juvenile hormone analogue and 20-hydroxyecdysone diminished the active K+ uptake, affecting the (Na+, K+)-pump and the other K+-pump(s) differently. In the presence of ouabain or at alkaline pH, mid-vitellogenic follicles developed in vitro rather normally up to the end of oogenesis whereas the other inhibitors as well as acidic pH blocked the course of development. The relevance of these findings for intracellular pH-regulation, osmoregulation, growth, membrane potentials, ionic currents, intrafollicular transport and other developmental processes is discussed.

Observations on the polarity of mutant Drosophila follicles lacking the oocyte

SummaryHomozygous females of the mutantsegalitarian andBicaudal-DR26produce 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.

Aberrant oogenesis in the patterning mutant dicephalic of Drosophila melanogaster: time-lapse recordings and volumetry in vitro

SummaryDrosophila females homozygous for the mutation dicephalic occasionally produce ovarian follicles with a nurse-cell cluster on each oocyte pole (dic follicles). Most dic follicles contain 15 nurse cells as in the normal follicle, but the total nurse-cell volume is larger in dic follicles; this is in keeping with the increase in DNA content recently described. However, the relative increase in oocyte volume during nurse-cell regression (from stage 10B onward) is not significantly larger in dic than in normal follicles. Time-lapse recordings in vitro show that, as a rule, both nurse cell clusters in a dic follicle export cytoplasm to the oocyte but nurse-cell regression remains incomplete at both poles and the persisting remnants of the nurse cells cause anomalies in chorion shape. The kinematics of cytoplasmic transfer are less aberrant at that oocyte pole which harbours the germinal vesicle. Possible links are discussed between these anomalies of oogenesis and the double-anterior embryonic patterns observed in the majority of developing dic eggs.