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1.
Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.  相似文献   

2.
All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (beta-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.  相似文献   

3.
When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.  相似文献   

4.
Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). We have previously demonstrated that dFmr1, the Drosophila ortholog of the fragile X mental retardation 1 gene, plays a role in the proper maintenance of germline stem cells in Drosophila ovary; however, the molecular mechanism behind this remains elusive. In this study, we used an immunoprecipitation assay to reveal that specific microRNAs (miRNAs), particularly the bantam miRNA (bantam), are physically associated with dFmrp in ovary. We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance. Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells. Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.  相似文献   

5.
Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.  相似文献   

6.
Theovarian tumorgene is required during both early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. We demonstrate that each of these phenotypes is associated with specific aberrations in actin distribution. In the earliest case,ovarian tumormutations cause actin filaments to accumulate ectopically in the fusome. This correlates with abnormal fusome morphology and arrested germ cell development in the germaria. Similarly,ovarian tumorfunction is required for the localization of actin that is essential for the maturation of ring canals. This defect gives rise to tumorous egg chambers in which germ cell numbers and morphology are profoundly aberrant. We also confirm thatovarian tumoris required for the formation of the nurse cell cytoplasmic actin array that is essential for the nonspecific transport of cytoplasmic contents to the oocyte during late oogenesis. Our data suggest that at this stageovarian tumorcontrols the site where actin filaments initiate. Taken together, these studies suggest that the diverseovarian tumormutant phenotypes derive from the mislocalization of actin filaments, indicating a role for this gene in organizing the female germline cytoskeleton, and that the misregulation of actin can have profound effects on germ cell division and differentiation.  相似文献   

7.
In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.  相似文献   

8.
Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.  相似文献   

9.
The PAR-1 kinase is required for the posterior localisation of the germline determinants in C. elegans and Drosophila, and localises to the posterior of the zygote and the oocyte in each case. We show that Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localisation within the oocyte at two stages of oogenesis. Finally, we show that PAR-1 localises on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte.  相似文献   

10.
In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.  相似文献   

11.
BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.  相似文献   

12.
Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell–generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell–like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.  相似文献   

13.
In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.  相似文献   

14.
15.
Lissencephaly is a severe congenital brain malformation resulting from incomplete neuronal migration. One causal gene, LIS1, is homologous to nudF, a gene required for nuclear migration in A. nidulans. We have characterized the Drosophila homolog of LIS1 (Lis1) and show that Lis1 is essential for fly development. Analysis of ovarian Lis1 mutant clones demonstrates that Lis1 is required in the germline for synchronized germline cell division, fusome integrity and oocyte differentiation. Abnormal packaging of the cysts was observed in Lis1 mutant clones. Our results indicate that LIS1 is important for cell division and differentiation and the function of the membrane cytoskeleton. They support the notion that LIS1 functions with the dynein complex to regulate nuclear migration or cell migration.  相似文献   

16.
RBBP6 (retinoblastoma binding protein 6, also known as PACT or P2P-R in humans) is a multi-domain protein that functions in multiple processes, such as mitosis, cell differentiation, and cell apoptosis. RBBP6 is evolutionarily conserved and is present in unicellular organisms to mammals. Studies of RBBP6 have mostly focused on its RB- and p53-binding domains, which are found exclusively in mammals. Here, we investigated the C. elegans homolog of RBBP6 to explore the functional roles of its other domains. We found that RBPL-1, the homolog of RBBP6 in C. elegans, is indispensable for worm development. RNAi silencing of rbpl-1 led to embryonic lethality, as well as defects in oocyte production and intestine development. rbpl-1 RNAi worms showed defects in germ cell proliferation, suggesting that RBPL-1 regulates mitosis. Moreover, RNAi silencing of rbpl-1 inhibited nutrient synthesis in the worm intestine. RBPL-1, as a nucleolus protein, was found to be expressed in diverse tissues and necessary for both germline and soma development. Using microarray analysis, we identified ≈700 genes whose expression levels were changed at least 10-fold in rbpl-1 worms. We propose that RBPL-1, like its yeast homolog, may regulate gene expression as an mRNA cleavage and polyadenylation factor. Taken together, the findings from this study reveal that RBPL-1 plays a pivotal role in C. elegans germline and soma development, suggesting that the functions of RBBP6 are conserved in diverse eukaryotic species.  相似文献   

17.
18.
Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.  相似文献   

19.
Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 µm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance.  相似文献   

20.
In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.  相似文献   

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