ABSTRACT
During Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi 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.
Subject(s)
Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Nuclear Proteins/metabolism , Oocytes/cytology , Oogenesis/physiology , RNA-Binding Proteins/metabolism , Animals , Body Patterning , Carrier Proteins/genetics , Cell Differentiation , Drosophila Proteins/genetics , Genes, Insect , Nuclear Proteins/genetics , Oocytes/metabolism , Phenotype , RNA-Binding Proteins/geneticsABSTRACT
A protein complex consisting of Mago Nashi and Tsunagi/Y14 is required to establish the major body axes and for the localization of primordial germ cell determinants during Drosophila melanogaster oogenesis. The Mago Nashi:Tsunagi/Y14 heterodimer also serves as the core of the exon junction complex (EJC), a multiprotein complex assembled on spliced mRNAs. In previous studies, reduced function alleles of mago nashi and tsunagi/Y14 were used to characterize the roles of the genes in oogenesis. Here, we investigated mago nashi and tsunagi/Y14 using null alleles and clonal analysis. Germline clones lacking mago nashi function divide but fail to differentiate. The mago nashi null germline stem cells produce clones over a period of at least 11 days, suggesting that mago nashi is not necessary for stem cell self-renewal. However, germline stem cells lacking tsunagi/Y14 function are indistinguishable from wild type. Additionally, in tsunagi/Y14 null germline cysts, centrosomes and oocyte-specific components fail to concentrate within a single cell and oocyte fate is not restricted to a single cell. Together, our results suggest not only that mago nashi is required for germline stem cell differentiation but that surprisingly mago nashi functions independently of tsunagi/Y14 in this process. On the other hand, Tsunagi/Y14 is essential for restricting oocyte fate to a single cell and may function with mago nashi in this process.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Germ Cells/cytology , Germ Cells/metabolism , Nuclear Proteins/metabolism , Oogenesis/physiology , RNA-Binding Proteins/metabolism , Animals , Animals, Genetically Modified , Cell Differentiation , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Female , Gene Expression Regulation, Developmental , Genes, Insect , Nuclear Proteins/genetics , Oocytes/cytology , Oocytes/metabolism , Oogenesis/genetics , RNA-Binding Proteins/geneticsABSTRACT
Vlad tepes (vlt(m651)) is one of only five "bloodless" zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in vlt(m651). Embryos homozygous for the vlt(m651) mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C --> T (Arg-339 --> Stop), in vlt(m651). The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in vlt(m651). These results show that the vlt(m651) mutation is a previously uncharacterized gata1 allele in the zebrafish. The vlt(m651) mutation sheds new light on Gata1 structure and function in vivo, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.
