During oogenesis, a germline stem cell divides forming a cyst of
During oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte. cells (cystocytes) interconnected by intercellular bridges, called ring canals (Bning, 1994; de Cuevas et al., 1997; Pepling et al., 1999). For example, in and occurs in the anterior tip of the ovary, in a region designated as the germarium (King, 1970; Spradling, 1993). Within the anterior tip of the germarium, a germline stem cell (GSC) divides, producing a replacement GSC and a cystoblast (Wong et al., 2005). GSCs and cystoblasts both have a spherical cytoplasmic membranous structure, the Praeruptorin B spectrosome (Deng and Lin, 1997; Lin et al., 1994). In subsequent cystoblast and cystocyte divisions, spectrosomes become branched structures (fusomes) extending through the ring canals into each cell of the cyst. Fusome material is partitioned unequally at each cyst-forming division, resulting in a 16-cell cyst with two cells (pro-oocytes) containing more fusome material than the other cells (de Cuevas and Spradling, 1998). One of the two pro-oocytes differentiates as the oocyte, while the other pro-oocyte and cells within the cyst become nurse cells. The fusomes consist of membrane skeletal proteins (e.g., Adducin-like protein, Ankyrin, -Spectrin and -Spectrin) and are associated with microtubule motors (e.g., Dynein heavy chain 64C and Klp61F), microtubule-associated proteins (e.g., Deadlock, Lis1, Orbit/Mast and Spectraplakin) Rabbit Polyclonal to OR8S1 and other fusome-interacting proteins (de Cuevas et al., 1996; Lin et al., 1994; Liu et al., 1999; Mth et al., 2003; McGrail and Hays, 1997; Petrella et al., 2007; R?per and Brown, 2004; Wehr et al., 2006; Wilson, 1999; Yue and Spradling, 1992). Mutations in genes encoding fusome components (e.g., -and (mRNA within the oocyte posterior pole is essential for abdominal segmentation and for determination of primordial germ cells (Ephrussi et al., 1991; Ephrussi and Lehmann, 1992). During oogenesis, Mago and Tsu/Y14 co-localize within the nuclei of germline and follicle cells. In the germarium, Mago and Tsu/Y14 also co-localize within the cytoplasm of germline cells in a pattern that is indistinguishable from BicD, Egl, and Orb, components necessary for oocyte differentiation (Parma et al., 2007). Germline clones generated by employing a reduced function allele of are similar phenotypically to null germline clones. Together, the co-localization of Mago and Tsu/Y14 within the cytoplasm of germarial germline cells and the similarity of the germline clonal phenotype of a reduced function allele and the null allele suggest that Mago and Tsu/Y14 function jointly to restrict oocyte fate to a single cell. Employing a shotgun proteomics approach, we identified at least 54 proteins that repeatedly co-immunoprecipitate (co-IP) with Mago (Bennett and Boswell, unpublished). One of the proteins is encoded by and we designated the encoded protein as Ranshi (Japanese for oocyte). Here we show that Ranshi co-IPs with Mago and Praeruptorin B Tsu/Y14 from ovarian extracts, indicating that the proteins form an oogenic complex. To gain an understanding of Ranshis role during oogenesis, we also characterized the phenotypes of ovaries derived from homozygous and hemizygous females. Our phenotypic analyses of mutant females reveal that in ovaries deficient for Ranshi, components required for oocyte differentiation accumulate within the presumptive oocyte but fail to localize within the posterior pole, indicating that Ranshi forms a complex with EJC components that influences the posterior pole localization/anchoring of oocyte differentiation factors. Materials and methods Take flight shares, culturing, immunolocalization and immunoprecipitation Standard methods were used for all crosses and culturing. Oogenic phases are relating to Spradling (1993). Lines utilized to study were explained previously (Parma et al., 2007). The following additional lines were used: (1) oogenic phenotypes are explained in Parma et. al. (2007). Ovaries were dissected from females 0-1 day time after eclosion. Immunoprecipitation and immunohistochemistry were Praeruptorin B performed as explained in earlier studies (Mohr et al., 2001; Parma et al., 2007). Immunoprecipitated proteins were prepared for mass spectrometry as explained by Andersen et. al. (2003) and the samples were analyzed by the University or college of Colorado, Mass Spectrometry, Central Analytical Lab. To determine healthy proteins, we utilized the Mascot system (Matrix Technology Inc.) to search the Country wide Center for Biotechnology Info and Flybase directories. Immunoblotting of co-immunoprecipitated healthy proteins and genetic connection studies were used to verify healthy proteins found by mass spectrometry. Transgenes We generated two genomic save constructs, p[Taq polymerase (Invitrogen). To facilitate cloning primers contained the restriction sites EcoRI and XhoI: FranshiXhoI 5-CTCCTCGAGCTGAAAGCCGAGATGAAACAGG-3, and RranshiEcoRI 5-CTCGAATTCCCGCGGATGCCTTTACGATAGA-3 and N8159-ranshi 5-CCTCGAGAGCTGTGCGACAAGTCCTTT-3 and L8159-ranshi 5-TGCGGCCGCAGCCCATGGACAAAACTCTG-3. The transgenes were confirmed.