ABSTRACT
Experiments in cultured cells with Ran-binding protein M (RanBPM) suggest that it links cell surface receptors and cell adhesion proteins. In this study, we undertake a genetic study of RanBPM function in the germline stem cell (GSC) niche of Drosophila melanogaster ovaries. We find that two RanBPM isoforms are produced from alternatively spliced transcripts, the longer of which is specifically enriched in the GSC niche, a cluster of somatic cells that physically anchors GSCs and expresses signals that maintain GSC fate. Loss of the long isoform from the niche causes defects in niche organization and cell size and increases the number of GSCs attached to the niche. In genetic mosaics for a null RanBPM allele, we find a strong bias for GSC attachment to mutant cap cells and observe abnormal accumulation of the adherens junction component Armadillo (beta-catenin) and the membrane skeletal protein Hu-li tai shao in mutant terminal filament cells. These results implicate RanBPM in the regulation of niche capacity and adhesion.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cytoskeletal Proteins/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/cytology , Nuclear Proteins/physiology , Ovum/cytology , Stem Cells/cytology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Alleles , Alternative Splicing , Amino Acid Motifs , Animals , Armadillo Domain Proteins/metabolism , Calmodulin-Binding Proteins/metabolism , Cell Adhesion/genetics , Cell Differentiation/genetics , Cell Proliferation , Cell Shape/genetics , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Mosaicism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Oogenesis , Ovary/metabolism , Ovum/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/physiology , Stem Cells/metabolism , Transcription Factors/metabolismABSTRACT
Germline stem cells (GSCs) in Drosophila are a valuable model to explore of how adult stem cells are regulated in vivo. Genetic dissection of this system has shown that stem cell fate is determined and maintained by the stem cell's somatic microenvironment or niche. In Drosophila gonads, the stem cell niche -- the cap cell cluster in females and the hub in males -- acts as a signaling center to recruit GSCs from among a small population of undifferentiated primordial germ cells (PGCs). Short-range signals from the niche specify and regulate stem cell fate by maintaining the undifferentiated state of the PGCs next to the niche. Germline cells that do not receive the niche signals because of their location assume the default fate and differentiate. Once GSCs are specified, adherens junctions maintain close association between the stem cells and their niche and help to orient stem cell division so that one daughter is displaced from the niche and differentiates. In females, stem cell fate depends on bone morphogenetic protein (BMP) signals from the cap cells; in males, hub cells express the cytokine-like ligand Unpaired, which activates the Janus kinase-signal transducers and activators of transcription (Jak-Stat) pathway in stem cells. Although the signaling pathways operating between the niche and stem cells are different, there are common general features in both males and females, including the arrangement of cell types, many of the genes used, and the logic of the system that maintains stem cell fate.
Subject(s)
Drosophila/cytology , Germ Cells/cytology , Stem Cells/cytology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Cell Movement , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Female , Germ Cells/metabolism , Male , Signal Transduction , Stem Cells/metabolism , Transcription, GeneticABSTRACT
The segmentation of the proximal-distal axis of the Drosophila melanogaster leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. We report here that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. We find that Serrate and Delta are ectopically expressed in dAP-2 mutant legs and that Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. We also find that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation.
Subject(s)
Calcium-Binding Proteins/metabolism , Digestive System Abnormalities/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Transcription Factor AP-2/genetics , Animals , Calcium-Binding Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Extremities/growth & development , Genes, Reporter , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Membrane Proteins/genetics , Models, Genetic , Mutation , Serrate-Jagged ProteinsABSTRACT
Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (VAS) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and VAS is a candidate translational activator in the pole plasm. VAS is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. Here, we used a biochemical approach to identify proteins that copurify with VAS in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (FAF), a pole plasm component [7], but one whose roles in posterior patterning and germ line specification have remained unclear. We present evidence that FAF interacts with VAS physically and reverses VAS ubiquitination, thereby stabilizing VAS in the pole plasm.
Subject(s)
Cytoplasmic Granules/metabolism , Endopeptidases/metabolism , Gene Expression Regulation, Developmental , Oocytes/metabolism , RNA Helicases/metabolism , Animals , Blotting, Western , DEAD-box RNA Helicases , Drosophila , Drosophila Proteins , Electrophoresis, Polyacrylamide Gel , Endopeptidases/isolation & purification , Precipitin Tests , RNA Helicases/isolation & purificationABSTRACT
We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.