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.

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.

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.

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.

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.

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.

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.

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.