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 Trl expression. 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.     

Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.     

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.     

Ovarian tumor expression is dependent on the functions of the somatic sex regulatory genes transformer-2 and doublesex

The doublesex-dependent sex regulatory pathway in Drosophila controls major aspects of somatic sexual differentiation, but its expression is not required in the X/X germline. Nevertheless, mutations in doublesex and in the genes that directly regulate its expression, transformer and transformer-2, disrupt early stages of oogenic differentiation to produce gonads containing immature germ cells. This indicates a critical, but uncharacterized, set of soma-germline interactions essential for oogenesis. In this paper, we examined the effects of mutations in transformer-2 on the expression and function of the germline-specific ovarian tumor gene. We demonstrated that in transformer-2 mutants, there was a marked reduction in the activity of the ovarian tumor promoter in the mutant germline. In addition, the phenotypic effects on the arrested germline could be partially suppressed by the simultaneous over-expression of both ovarian tumor and a second germline gene, Sex-lethal. This differs from transformer mutations, in which the over-expression of ovarian tumor alone is sufficient for a similar improvement in germline differentiation. In contrast to transformer-2, doublesex activity was not required for ovarian tumor promoter activity and we found indirect evidence that the doublesex male-specific function might have a negative regulatory effect. These data indicate that the components of the genetic pathway regulating somatic sexual differentiation have specific and differential effects on germline gene activity.     

fringe and Notch specify polar cell fate during Drosophila oogenesis

fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles.     

piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells

piwi represents the first class of genes known to be required for stem cell self-renewal in diverse organisms. In the Drosophila ovary, piwi is required in somatic signaling cells to maintain germline stem cells. Here we show that piwi encodes a novel nucleoplasmic protein present in both somatic and germline cells, with the highly conserved C-terminal region essential for its function. Removing PIWI protein from single germline stem cells significantly decreases the rate of their division. This suggests that PIWI has a second role as a cell-autonomous promoter of germline stem cell division. Consistent with its dual function, over-expression of piwi in somatic cells causes an increase both in the number of germline stem cells and the rate of their division. Thus, PIWI is a key regulator of stem cell division - its somatic expression modulates the number of germline stem cells and the rate of their division, while its germline expression also contributes to promoting stem cell division in a cell-autonomous manner.     

A gene trap mutation of a murine homolog of the Drosophila stem cell factor Pumilio results in smaller testes but does not affect litter size or fertility

Members of the Pumilio (also called PUF) gene family belong to a class of highly conserved developmental regulators that are present in both flies and humans. Much is known about the function of Pumilio genes in invertebrate development, in particular their role as stem cell factors required for maintenance and/or self-renewal of germline stem cells in Drosophila and Caenorhabditis elegans. It remains unknown whether Pumilio genes are also required for development in mammals; however, several lines of evidence suggest similar functions based on extensive sequence homology, similar RNA-binding properties to their invertebrate counterparts and well-documented interactions with germ cell factors required for fertility. Here we report characterization of a gene trap mutation that disrupts the mouse Pumilio-2 (Pum2) gene. Our data confirm that Pumilio-2 is expressed most abundantly in germ cells with the highest expression in undifferentiated gonocytes and spermatogonia. Furthermore, the mutation in Pum2 results in significantly smaller testes although the mutants are otherwise viable and fertile. In addition, we observed no stronger reproductive defects on a genetic background homozygous for a Pum2 null mutation and heterozygous for a Dazl mutation than Pum2 mutant alone. Thus, as in C. elegans where single members of the Pumilio gene family are dispensable for reproductive development and viability, this individual member of the Pumilio gene family in mice is also not essential for reproduction or viability.     

The Drosophila ovary: an active stem cell community

Only a small number of cells in adult tissues (the stem cells) possess the ability to self-renew at every cell division,while producing differentiating daughter cells to maintain tissue homeostasis for an organism's lifetime.The Drosophilaovary harbors three different types of stem cell populations (germline stem cell (GSC),somatic stem cell (SSC) andescort stem cell (ESC)) located in a simple anatomical structure known as germarium,rendering it one of the best modelsystems for studying stem cell biology due to reliable stem cell identification and available sophisticated genetic toolsfor manipulating gene functions.Particularly,the niche for the GSC is among the first and best studied ones,and studieson the GSC and its niche have made many unique contributions to a better understanding of relationships between stemcells and their niche.So far,both the GSC and the SSC have been shown to be regulated by extrinsic factors originatingfrom their niche and intrinsic factors functioning within.Multiple signaling pathways are required for controlling GSCand SSC self-renewal and differentiation,which provide unique opportunities to investigate how multiple signals fromthe niche are interpreted in the stem cell.Since the Drosophila ovary contains three types of stem cells,it also providesoutstanding opportunities to study how multiple stem cells in a given tissue work collaboratively to contribute to tissuefunction and maintenance.This review highlights recent major advances in studying Drosophila ovarian stem cells andalso discusses future directions and challenges.     

Evidence for Drosophila P element transposase activity in mammalian cells and yeast

Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.     

Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans

Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1-knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.     

Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

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.