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.     

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).     

Oocyte and embryonic cytoskeletal defects caused by mutations in the <Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">swallow</Emphasis> gene

The maternal effect gene swallow ( swa) of Drosophila is required for bicoid and htsN4 mRNA localization during oogenesis. Swallow is also required for additional, poorly understood, functions in early embryogenesis. We have examined the cytoskeleton in swa mutant oocytes and embryos by immunocytochemistry and confocal microscopy. Mid- and late-stage swaoocytes have defective cytoplasmic actin networks. Stage-10 oocytes have solid actin clumps and hollow actin spheres in the subcortical layer, and late-stage oocytes have uniformly distributed hollow actin spheres in the subcortical layer and in deeper cytoplasm. Swa preblastoderm embryos have uneven and irregularly distributed actin at the cortex, and defective subcortical actin networks that contain hollow and solid spheres. In swa syncytial blastoderm embryos, the abnormal actin cytoskeleton is associated with defects in nuclear distribution, migration and cleavage. Actin cytoskeletal defects correlate with spindle defects, suggesting that the abnormal organization of the actin cytoskeleton allows interaction of mitotic spindles, which induces defective nuclear divisions and loss of nuclei from the surface of the embryo.     

The Drosophila RNA-binding protein Lark is required for the organization of the actin cytoskeleton and Hu-li tai shao localization during oogenesis

Elimination of maternal expression of the Drosophila RNA-binding protein Lark results in female sterility. Here we show that this is due to a requirement during oogenesis. Developing oocytes from lark(1) germline clones (GLCs) are often smaller than normal due to defects in nurse cell cytoplasmic "dumping." Late-stage egg chambers from lark(1) GLCs contain low levels of cortical and ring canal associated actin and completely lack nurse cell cytoplasmic F-actin bundles, suggesting the "dumping" phenotype is due to a defect in the actin cytoskeleton. Localization of Hu-li tai shao (Hts) protein, a component of ring canals, is also disrupted in these mutants. In addition to the dumpless phenotype, we observed a buildup of late-stage egg chambers, a phenotype that correlates with the decrease in egg-laying observed in the mutants. We postulate that this phenotype is due to defects in the cytoskeletal integrity of eggs since retained and oviposited eggs are fragile and often deflated. These mutant phenotypes are likely due to disruption of an RNA-binding function of Lark as similar phenotypes were observed in flies carrying specific RNA-binding domain mutations. We propose that Lark functions during oogenesis as an RNA-binding protein, regulating mRNAs required for nurse cell transport or apoptosis.     

Intercellular cytoplasm transport during oogenesis of the moth midge, Tinearia alternata Say (Diptera: Psychodidae)

Polytrophic ovaries of the nematocerous dipteran, Tinearia alternata Say consists of several developmentally synchronized ovarioles each housing only one functional egg chamber with 15 nurse cells and an oocyte. At the early stages of previtellogenesis the nurse cells become polyploid and synthetically active. Their nuclei contain polytene chromosomes and prominent nucleoli. With the advance of previtellogenic growth the nurse cell cytoplasm is loaded with the growing number of ribosomes and contain perinuclear nuage material, mitochondria, electron dense bodies and aggregations of endoplasmic reticulum. All these organelles are transported into the oocyte thanks to the massive and rapid flow of the nurse cell cytoplasmic contents. Nurse cell-oocyte transport is mediated by actin cytoskeleton. Prior to the rapid cytoplasm transfer, F-actin network is associated with the nurse cell membranes while tiny bundles of microfilaments form actin baskets connected with ring canals. Nurse cells in Tinearia lack an extensive scaffold of radially oriented, F-actin bundles (cables) that would tether their nuclei in place, thus preventing ring canals from plugging. The way the nuclei are anchored to their central positions within the cells remains unclear. Towards the final stages of oogenesis nurse cells are almost devoid of cytoplasm and degenerate. Although their nuclei undergo dramatic morphological transformations, typical hallmarks of apoptotic pathway could not be clearly observed. Rapid ooplasmic streaming does not occur.     

Oogenesis in Hydra: nurse cells transfer cytoplasm directly to the growing oocyte

Oogenesis in Hydra occurs in so-called egg patches containing several thousand germ cells. Only one oocyte is formed per egg patch; the remaining germ cells differentiate as nurse cells. Whether and how nurse cells contribute cytoplasm to the developing oocyte has been unclear. We have used tissue maceration to characterize the differentiation of oocytes and nurse cells in developing egg patches. We show that nurse cells decrease in size at the same time that developing oocytes increase dramatically in volume. Nurse cells are also tightly attached to oocytes at this stage and confocal images of egg patches stained with the fluorescent membrane dye FM 4-64 clearly show large gaps (10 microm) in the cell membranes separating nurse cells from the developing oocyte. We conclude that nurse cells directly transfer cytoplasm to the developing oocyte. Following this transfer of cytoplasm, nurse cells undergo apoptosis and are phagocytosed by the oocyte. These results demonstrate that basic mechanisms of alimentary oogenesis typical of Caenorhabditis and Drosophila are already present in the early metazoan Hydra.     

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee . We named the allele chickadee^bin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadee^{wc57 and WF57} (2). In addition, the egg chamber of chickadee^bin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.     

Ultrastructure and function of nurse cells in phthirapterans. Possible function of ramified nurse cell nuclei in the cytoplasm transfer

The structure of nurse cells as well as the distribution of cytoskeletal elements (actin filaments, microtubules) in three representatives of phthirapterans: the pig louse, Haematopinus suis (Anoplura) and bird lice, Eomenacanthus stramineus, Columbicola columbae (Mallophaga) were investigated. All three species have polytrophic-meroistic ovaries which means that each oocyte remains connected with a group of nurse cells via specialized cytoplasmic canals-intercellular bridges (ring canals). Throughout vitellogenesis, various macromolecules as well as organelles (mitochondria, endoplasmic reticulum vesicles, ribosomes) are transferred from the nurse cells to the oocyte. During this flow, the nurse cell nuclei do not enter the oocyte and are retained in the cell centers. In holometabolous insects (e.g. Drosophila, hymenopterans), the central position of nurse cell nuclei is maintained by cytoskeletal elements (actin filaments or microtubules). In the investigated species, the nurse cells are equipped with large, highly extended (irregularly lobed) nuclei. The inner nuclear membrane is lined with a relatively thick layer of nuclear lamina. Ultrastructural analysis and staining with rhodamine-labeled phalloidin revealed that the nurse cell cytoskeleton is poorly developed and represented only by: (1) single microtubules in the perinuclear cytoplasm; and (2) the F-actin layer in the cortical cytoplasm. In the light of this, we postulate that in phthirapterans the position of nurse cell nuclei during the cytoplasm transfer is maintained not by the cytoskeletal elements, but by a largely extended shape of the nuclei (i.e. their elongated extensions).     

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.     

The Drosophila nucleoporin gene nup154 is required for correct microfilament dynamics and cell death during oogenesis

The Drosophila nucleoporin gene nup154 is required in both male and female germline for successful gametogenesis. Mutant flies lack differentiated sperm and lay abnormal eggs. We demonstrated that the egg phenotype was associated with specific alterations of the actin cytoskeleton at different stages of oogenesis. Actually, mutant egg chambers displayed an abnormal organization of both subcortical microfilaments and cytoplasmic actin bundles, that led to defective nurse cell dumping. TUNEL analysis also showed that the dumpless phenotype was associated with delayed apoptosis. The nup154 gene product was localized by conventional immunofluorescence microscopy to the nuclear envelope in a distinct punctuate pattern, characteristic of nuclear pore complex components. TEM analysis revealed that the protein was mainly distributed along filamentous structures that extended radially on the nuclear side of the pore, suggesting that Nup154 could be an integral component of the basket filaments associated with the nuclear pore complexes. We propose that Nup154 is necessary for correct nuclear pore complex functions and that the proper regulation of the actin cytoskeleton dynamics strongly relies upon nuclear pore integrity.     

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.     

On the roles of the Drosophila KASH domain proteins Msp-300 and Klarsicht

KASH (Klarsicht, Anc-1, Syne-1 homology) domain-containing proteins anchor the nucleus to the actin cytoskeleton or to microtubules. KASH proteins thus play pivotal roles in a variety of developmental processes where nuclear positioning is critical. Two KASH proteins have been identified in Drosophila: Muscle-specific protein-300 (Msp-300) and Klarsicht (Klar). Msp-300 anchors nuclei to actin, and has been reported to be essential for positioning of nurse cell nuclei during oogenesis, and thus production of mature ooctyes. Klar is required for positioning of photoreceptor and cone cell nuclei in the developing eye, which is critical for proper eye morphology. Here, we asked whether KASH domain-containing forms of Msp-300 are required for nuclear positioning in the eye, and we found that they are not. Moreover, in the course of this work, we discovered that contrary to previous reports, KASH domain-containing forms of Msp-300 are not required for viability, nor for oogenesis. However, we did find that Msp-300 has a function in egg laying, normally redundant with a function of Klar.     

On the roles of the Drosophila KASH domain proteins Msp-300 and Klarsicht

KASH (Klarsicht, Anc-1, Syne-1 homology) domain-containing proteins anchor the nucleus to the actin cytoskeleton or to microtubules. KASH proteins thus play pivotal roles in a variety of developmental processes where nuclear positioning is critical. Two KASH proteins have been identified in Drosophila: Muscle-specific protein-300 (Msp-300) and Klarsicht (Klar). Msp-300 anchors nuclei to actin, and has been reported to be essential for positioning of nurse cell nuclei during oogenesis, and thus production of mature ooctyes. Klar is required for positioning of photoreceptor and cone cell nuclei in the developing eye, which is critical for proper eye morphology. Here, we asked whether KASH domain-containing forms of Msp-300 are required for nuclear positioning in the eye, and we found that they are not. Moreover, in the course of this work, we discovered that contrary to previous reports, KASH domain-containing forms of Msp-300 are not required for viability, nor for oogenesis. However, we did find that Msp-300 has a function in egg laying, normally redundant with a function of Klar.     

Multiple effects of colchicine on oogenesis in Drosophila: Induced sterility and switch of potential oocyte to nurse-cell developmental pathway

Adult female fruitflies exposed to colchicine admixed to the culture medium show a series of dosage-related abnormalities that affect oogenesis and may induce sterility. Among the effects observed were decreased fecundity and hatchability of laid eggs, formation of oocytes lacking chorionic appendages, abnormal distribution and diminution in number of yolk spheres, inhibition of oocyte growth and abnormally located oocyte nuclei. Potentially the most significant effect was the development of egg chambers which contained the normal complement of 16 cells but in which all the cells had the nuclear morphology of nurse cells. The approach provides for the first time an experimental means to divert a potential oocyte into the developmental pathway of the nurse cell in a wild-type fly, and hence should be helpful in the elucidation of factors which control oocyte and nurse cell differentiation. In addition, the results serve to expand the usefulness of oogenesis in Drosophila as a model system for the evaluation of drug-induced metabolic-morphologic abnormalities.     

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.     

The cytoskeleton organizes germ nuclei with divergent fates and asynchronous cycles in a common cytoplasm during oogenesis in the chordate Oikopleura

Germline cysts are conserved structures in which cells initiating meiosis are interconnected by ring canals. In many species, the cyst phase is of limited duration, but the chordate, Oikopleura, maintains it throughout prophase I as a unique cell, the coenocyst. We show that despite sharing one common cytoplasm with meiotic and nurse nuclei evenly distributed in a 1:1 ratio, both entry into meiosis and subsequent endocycles of nurse nuclei were asynchronous. Coenocyst cytoskeletal elements played central roles as oogenesis progressed from a syncytial state of indistinguishable germ nuclei, to a final arrangement where the common cytoplasm had been equally partitioned into resolved, mature oocytes. During chromosomal bouquet formation in zygotene, nuclear pore complexes clustered and anchored meiotic nuclei to the coenocyst F-actin network opposite ring canals, polarizing oocytes early in prophase I. F-actin synthesis was required for oocyte growth but movement of cytoplasmic organelles into oocytes did not require cargo transport along colchicine-sensitive microtubules. Instead, microtubules maintained nurse nuclei on the F-actin scaffold and prevented their entry into growing oocytes. Finally, it was possible to both decouple meiotic progression from cellular mechanisms governing oocyte growth, and to advance the timing of oocyte growth in response to external cues.     

Monoclonal antibodies against Drosophila ovaries: their reaction with ovarian and embryonic antigens

A library of monoclonal antibodies (MAbs) against Drosophila ovarian antigens was established. Each of the MAbs was characterized by its immunohistochemical binding pattern to sections from egg chambers at various stages of oogenesis. Sixteen of the 18 MAbs were found to bind to antigens in mature oocytes. Among the 16 antigens, two were also located in cytoplasm of cell types in the egg chamber other than the oocyte, at all stages of oogenesis. Four made their appearance in nurse cell cytoplasm at mid-vitellogenic stages and shifted to oocyte cytoplasm at a later stage, and ten appeared at the vitellogenic stage and confined their distribution to oocyte cytoplasm. All these antigens were distributed evenly in cytoplasm of mature oocytes. However, some of these antigens were noticed to change their distribution during early embryogenesis as to be localized in a specific region of embryos.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Accessory nuclei in insect oogenesis: in search of the function of enigmatic organelles

This review compiles present knowledge of the structure and molecular composition of the enigmatic cytoplasmic organelles called accessory nuclei. Most typically, they are found in the perinuclear cytoplasm in oocytes of insects and several other invertebrates. Accessory nuclei originate by budding of the oocyte nucleus (germinal vesicle) and are surrounded by an envelope identical to the nuclear envelope. They contain one or several dense inclusions called pseudonucleoli immersed in a translucent ground substance or matrix. Comparative analysis of the morphology, molecular composition and the ultimate fate of accessory nuclei and their inclusions revealed that there are two basic types of these organelles in insect oocytes. In mallophagans, accessory nuclei are associated with the oocyte nucleus throughout entire oogenesis and at least some of them are connected to the germinal vesicle by slender stems. Each accessory nucleus contains a single, dense, RNA-positive inclusion which is likely to correspond to a nucleolus. In hymenopterans, accessory nuclei initially surround the germinal vesicle but during oogenesis they separate from it and migrate toward the peripheral ooplasm. Within the accessory nucleus matrix usually two distinct inclusions develop. One of these is perfectly spherical, contains coilin and small nuclear ribonucleoproteins and is homologous to the Cajal body. In the light of recent discoveries, we discuss the role accessory nuclei play in insect oogenesis and early 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.