Drosophila germline stem cells for in vitro analyses of PIWI-mediated RNAi.

The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the maintenance and expansion of GSCs in an established cell line (fGS/OSS) and an in situ hybridization method for analyzing piwi.

Stable transformation and cloning mediated by piggyBac vector and RNA interference knockdown of Drosophila ovarian cell line.

An in vitro study is a powerful method for elucidating gene functions in cellular and developmental events. However, until date, no reliable in vitro transformation, cloning, or knockdown system has been reported for Drosophila cells, with the exception of S2 and Kc cells. In this study, we demonstrated that the piggyBac vector stably integrates donor DNA into ovarian somatic sheets derived from follicle stem cells. The transformed ovarian somatic sheet cells were easily cloned with a new piggyBac selection vector carrying enhanced green fluorescent protein and dihydrofolate reductase genes, egfp, and dhfr, respectively, in culture media containing methotrexate, an inhibitor of DNA synthesis. Donor egfp continued to be expressed at a high level in long-term culture. Furthermore, the translation of donor egfp was inhibited by treatment with double-stranded RNA derived from the target gene. The transfection and cloning methods mediated by the piggyBac vector would thus be useful for future analyses of gene functions in OSS cells and possibly be applicable to other Drosophila cell lines.

BMP and Hh signaling affects primordial germ cell division in Drosophila.

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.

Culturing ovarian somatic and germline stem cells of Drosophila.

This unit describes how to collect, culture, and establish stable cell lines of ovarian somatic and germline stem cells of Drosophila. We also describe a protocol for culturing embryonic cells that overexpress growth factors, which serve as a source for conditioned medium.

Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line.

Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway.

Autonomous differentiation of Drosophila spermatogonia in vitro.

Spermatogenesis is a complex process that produces functional sperm by establishing male germline stem cells (mGSCs) in adult testes. To study Drosophila spermatogenesis in vitro, we examined various culture conditions of spermatogonia. Spermatogonia from larval testes began to differentiate soon after culture, whereas mGSCs did not undergo self-renewal division. Strikingly, 16-cell spermatogonia from early and late larval testes differentiated into motile spermatids autonomously. Furthermore, individual spermatogonia developed into motile spermatids even after mechanical dissociation from encapsulating cyst cells. This is the first study to report that spermatogonia in larval testes retain the ability to differentiate into spermatids in the absence of gonadal tissue. Our in vitro system should provide an excellent opportunity to study spermatogenesis in detail and apply genetic manipulation.

Wingless signaling initiates mitosis of primordial germ cells during development in Drosophila.

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, we examined the effects of various growth factors on the proliferation of PGCs. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/beta-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner.

Establishment of stable cell lines of Drosophila germ-line stem cells.

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam(-) GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma.

Ovarian cystocytes can repopulate the embryonic germ line and produce functional gametes.

Ovarian tumors are formed either in the absence of Bam (bag-of-marbles) in germ-line cells or the overexpression of Dpp (decapentaplegic) in ovarian somatic cells. These tumor cells contain spectrosomes characteristic of ovarian germ-line stem cells and the immediate descendents called cystoblasts. We show that pole cells can successfully populate the gonad after transplantation to the dorsal mesoderm of host embryos following germ-band extension. By using this approach, we demonstrate that bam- cells can populate the gonad and become established as germ-line stem cells. Tumor cells containing the wild-type bam gene under heat shock transcriptional control are able to produce functional oocytes. Thus, stem cells/cystoblasts of the adult ovary are capable of forming stem cells in the embryonic ovary and recapitulating the development of the female germ line.

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster.

Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1-33.8±0.7 forgs(1)N26 and 1-39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.