Germline Clones Research Articles

A Germline Clone Screen on the X Chromosome Reveals Novel Meiotic Mutants in Drosophila melanogaster

A Germline Clone Screen on the X Chromosome Reveals Novel Meiotic Mutants in Drosophila melanogaster

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

Liquid facets-Related (lqfR) Is Required for Egg Chamber Morphogenesis during Drosophila Oogenesis

Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR) is an Epsin N-terminal homology (ENTH) domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR). LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis.

Traction of neuronal migration by polarized adhesion

Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton [1]. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro [2, 3]. CP190 has an essential function in the nucleus as a chromatin insulator [4], but centrosomes and MTs appear unperturbed in Cp190 mutants [4, 5]. Thus, the centrosomal function of CP190, if any, is unclear. Here, we examine the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos.

RpL10A regulates oogenesis progression in the banana prawn Fenneropenaeus merguiensis and Drosophila melanogaster

To develop banana prawn ( Fenneropenaeus merguiensis) aquaculture, the mechanism of ovarian maturation is under investigation. In a previous study, we reported the RpL10A protein as an ovarian maturation stimulator. To further investigate the function of RpL10A, we turned to the fruit fly ( Drosophila melanogaster) to take advantage of the genetic tools available. Here, we elucidate the expression and function of RpL10A in the D. melanogaster ovary. RpL10A is expressed in the cytoplasm of both nurse and follicle cells throughout oogenesis. While shrimp have one RpL10A gene, D. melanogaster has two genes, RpL10Aa and RpL10Ab. RpL10Ab displays more similarity with shrimp RpL10A and was further investigated. RpL10Ab homozygous mutants are lethal and germline clone analysis showed that RpL10Ab is an essential gene in oogenesis. Moreover, RpL10Ab − germline clones resulted in premature death of the follicle cells. This phenotype is reminiscent of some insulin pathway mutants, suggesting that RpL10Ab may be involved in the insulin signaling pathway. In addition, RpL10Ab − follicle cells showed abnormal nuclei and membranes. Shrimp RpL10A rescued RpL10Ab homozygous mutants, revealing their functional conservation. Surprisingly, we found cell death in multiple tissues when RpL10A was over-expressed, suggesting that proper RpL10A levels are important. This research reveals novel findings about the role of RpL10A during oogenesis and may, in the future, lead to new approaches to stimulate ovarian development in shrimp.

Loss of the extraproteasomal ubiquitin receptor Rings lost impairs ring canal growth in Drosophila oogenesis

In Drosophila melanogaster oogenesis, there are 16 germline cells that form a cyst and stay connected to each other by ring canals. Ring canals allow the cytoplasmic transport of proteins, messenger ribonucleic acids, and yolk components from the nurse cells into the oocyte. In this paper, we describe the protein Rings lost (Rngo) and show that it is required for ring canal growth in germline cysts. rngo is an essential gene, and germline clones of a rngo-null allele show defects in ovary development, including mislocalization of ring canal proteins and fusion of germline cells. Rngo appears to be a ubiquitin receptor that possesses a ubiquitin-like domain, a ubiquitin-associated domain, and a retroviral-like aspartate protease (RVP) domain. Rngo binds to ubiquitin and to the 26S proteasome and colocalizes with both in germline cells, and its RVP domain is required for dimerization of Rngo and for its function in vivo. Our results thus show, for the first time, a function for a ubiquitin receptor in Drosophila development.

Localization and Activation of the Drosophila Protease Easter Require the ER-Resident Saposin-like Protein Seele

Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity.

Sec5, a member of the exocyst complex, mediatesDrosophilaembryo cellularization

Cellularization of the Drosophila embryo is the process by which a syncytium of approximately 6000 nuclei is subdivided into discrete cells. In order to individualize the cells, massive membrane addition needs to occur by a process that is not fully understood. The exocyst complex is required for some, but not all, forms of exocytosis and plays a role in directing vesicles to appropriate domains of the plasma membrane. Sec5 is a central component of this complex and we here report the isolation of a new allele of sec5 that has a temperature-sensitive phenotype. Using this allele, we investigated whether the exocyst complex is required for cellularization. Embryos from germline clones of the sec5(ts1) allele progress normally through cycle 13. At cellularization, however, cleavage furrows do not invaginate between nuclei and consequently cells do not form. A zygotically translated membrane protein, Neurotactin, is not inserted into the plasma membrane and instead accumulates in cytoplasmic puncta. During cellularization, Sec5 becomes concentrated at the apical end of the lateral membranes, which is likely to be the major site of membrane addition. Subsequently, Sec5 concentrates at the sub-apical complex, indicating a role for Sec5 in the polarized epithelium. Thus, the exocyst is necessary for, and is likely to direct, the polarized addition of new membrane during this form of cytokinesis.

Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila

Ca 2+ signaling pathways play important roles to complete meiosis from metaphase II arrest in vertebrate oocytes. However, less is known about the molecular mechanism of completion of meiosis in Drosophila females. Here, we provide direct evidence that calcineurin, a Ca 2+/calmodulin (CaM)-dependent phosphatase, is essential for meiotic progression beyond metaphase I in Drosophila oocytes. Oocytes from germline clones lacking CanB2, a calcineurin regulatory subunit B, failed to complete meiosis after egg activation, and laid eggs exhibited a meiotic arrested anaphase I chromosome configuration. Genetic analyses suggest that calcineurin activity is regulated by Sarah (Sra), a family member of regulators of calcineurin (RCANs), through a Sra phosphorylation-dependent mechanism. Our results support a view in which the phosphorylation of Sra not only acts to relieve the inhibitory effects of Sra, but also acts to activate calcineurin, thus explaining the role of RCAN proteins as positive regulators of calcineurin.

Genetic and Proteomic Evidence for Roles of Drosophila SUMO in Cell Cycle Control, Ras Signaling, and Early Pattern Formation

SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body–P body pathway

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, we show that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. We previously found that SMN-containing U bodies invariably associate with P bodies (Liu, J. L., and Gall, J. G. (2007). U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Proc. Natl. Acad. Sci. U. S. A. 104, 11655–11659.). Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, our results suggest that SMN is required for the functional integrity of the U body–P body pathway, which in turn is important for maintaining proper nuclear architecture.

Intramolecular Interactions Between the Src Homology 3 Guanylate Kinase Domains of Discs Large Regulate Its Function in Asymmetric Cell Division

Membrane-associated guanylate kinases (MAGUKs) regulate the formation and function of molecular assemblies at specialized regions of the membrane. Allosteric regulation of an intramolecular interaction between the Src homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to play a central role in regulating MAGUK function. Here we show that a mutant of the Drosophila MAGUK Discs large (Dlg), dlg(sw), encodes a form of Dlg that disrupts the intramolecular association while leaving the SH3 and GK domains intact, providing an excellent model system to assess the role of the SH3-GK intramolecular interaction in MAGUK function. Analysis of asymmetric cell division of maternal-zygotic dlg(sw) embryonic neuroblasts demonstrates that the intramolecular interaction is not required for Dlg localization but is necessary for cell fate determinant segregation to the basal cortex and mitotic spindle alignment with the cortical polarity axis. These defects ultimately result in improper patterning of the embryonic central nervous system. Furthermore, we demonstrate that the sw mutation of Dlg results in unregulated complex assembly as assessed by GukHolder association with the SH3-GK versus PDZ-SH3-GK modules of Dlg(sw). From these studies, we conclude that allosteric regulation of the SH3-GK intramolecular interaction is required for regulation of MAGUK function in asymmetric cell division, possibly through regulation of complex assembly.

Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

BackgroundProtein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.ResultsIn the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.ConclusionBased on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.

SAP18 Promotes Krüppel-dependent Transcriptional Repression by Enhancer-specific Histone Deacetylation

Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.

Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster

Hsp60C gene of Drosophila melanogaster shows a dynamic spatiotemporal expression during oogenesis and seems to contribute bulk of the Hsp60 family proteins in ovarioles. Hsp60 distribution overlaps with that of F-actin-rich membranes/structures in follicle, nurse, and egg cells throughout oogenesis. Skeletal muscle fibers associated with ovarioles and in other parts of the body show patterned location of Hsp60 in A-bands. During stages 11-12, Hsp60 accumulates at junctions of nurse cells and oocyte, where a new microtubule organizing center is known to develop. A recessive hypomorph allele, Hsp60C1 causes complete sterility of the rare surviving homozygous adults. Their egg chambers show very little Hsp60C transcripts or Hsp60 protein. Beginning at stages 6-7, Hsp60C1 chambers show a disorganized follicle cell layer with poor cell adhesion in addition to abnormal organization of F-actin and other cytoskeletal structures in follicle, nurse, and egg cells. Additionally, expression and localizations of Hrb98DE, Squid, and Gurken proteins in nurse cells and oocyte are also severely affected. Hsp60C1 homozygous follicle cell clones in Hsp60C1/+ ovarioles show disruptions in follicle epithelial and cytoskeleton arrangements. Likewise, egg chambers with Hsp60C1 homozygous germline clones in Hsp60C1/+ flies show abnormal oogenesis. Our results provide the first evidence for an essential role of Hsp60C in Drosophila oogenesis, especially in organization and maintenance of cytoskeletal and cell adhesion components.

Cop9 signalosome subunit 8 (CSN8) is essential for Drosophila development

The COP9 signalosome (CSN) is a multisubunit regulator highly conserved in evolution. We show here that CSN subunit 8 (CSN8) is essential for Drosophila development. CSN8 is maternally contributed and present throughout development. Null mutants generated in this study are larval lethal, showing phenotypes associated with mutations in either CSN4 (molting defects) or CSN5 (melanotic tumors). Analysis of mitotic and germ-line csn8(null) clones revealed the requirement of CSN8 for multiple developmental processes. The germ-line clones arrested at mid-oogenesis, while the mitotic clones led to deformed adult eyes or wings. CSN8 is present exclusively as part of the CSN holo-complex, and lack of CSN8 in the mutants leads to CSN instability. Consistent with this, Cullin deneddylation is impaired in the csn8(null) mutants.

Motility Screen Identifies Drosophila IGF-II mRNA-Binding Protein—Zipcode-Binding Protein Acting in Oogenesis and Synaptogenesis

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.

Involvement of co-repressors Groucho and CtBP in the regulation of single-minded in Drosophila

Dorso-ventral patterning results in the establishment of the two germ layers in the Drosophila embryo, mesoderm and mesectoderm, that are separated by a strip of cells giving rise to the mesectoderm and eventually to the ventral midline. The mesectoderm is specified by the expression of single-minded (sim) which is activated through the concerted action of Dorsal and Twist in addition to a Notch signal. In the mesoderm, sim is repressed by Snail together with the co-repressor C-terminal binding protein (CtBP). Here, we address the involvement of the two co-repressors CtBP and Groucho (Gro) in repression of sim in the neuroectoderm. It was shown earlier that sim is restricted in the neuroectoderm with help of Suppressor of Hairless [Su(H)] and Hairless. Using the female sterile technique, we generated germ line clones deficient for Gro, CtBP or Hairless and assayed sim mRNA relative to snail mRNA expression. We show that sim repression requires both co-repressors Gro and CtBP to be fully repressed in the neuroectoderm, suggesting that a repression complex is assembled including Su(H) and Hairless as was shown for other Notch target genes before. Moreover, our work implies that Gro is important for the repression of sim specifically within the mesoderm anlagen, indicating that Snail and CtBP are insufficient to entirely silence sim in this germ layer.

Post-transcriptional repression of theDrosophilamidkine and pleiotrophin homologmipleby HOW is essential for correct mesoderm spreading

The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, we implemented a series of selection methods on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos, and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesoderm-spreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple (a Drosophila midkine and pleiotrophin heparin-binding growth factor), in the mesoderm led to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE-positive cells, which are responsive to receptor tyrosine kinase (RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, our analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis.

CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory

DNA synthesis during S-phase and upon DNA repair is accompanied by chromatin assembly. The chromatin assembly factor CAF-1 has been biochemically well-characterized to deposit histones onto newly synthesized DNA. To gain insights into the in vivo functions of CAF-1 in Drosophila, we generated null mutants of the largest subunit of dCAF-1, dCAF-1-p180. We show that, unlike CAF-1 mutant yeast, dCAF-1-p180 mutant flies are hemizygous lethal. Removal of maternal dCAF-1-p180 activity by germline clones blocks oogenesis. Tissue-specific deletion of dCAF-1-p180 in the eye primordia disrupts eye development. In addition, reduction of dCAF-1-p180 activity suppresses gene silencing at heterochromatin and antagonizes Polycomb-mediated cell fate determination. Furthermore, heterozygous dCAF-1-p180 mutant flies display an increased sensitivity to γ-irradiation and a reduced efficiency in recombinational double strand break (DSB) repair. Our experiments also show that human hCAF-1-p150 can rescue the dCAF-1-p180 mutant flies, demonstrating a functional conservation of eukaryotic CAF-1 activities in vivo. Together, our results establish that dCAF-1-p180 is an essential gene for Drosophila development and further underscore the importance of dCAF-1 in regulating gene expression and DNA repair in vivo.