The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular and genetic description of a new hypomorphic mutation of Trithorax-like gene and analysis of its effect on Drosophila melanogaster oogenesis

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional protein GAGA involved in many cellular processes. We have isolated and described a new hypomorphic mutation of The Trl gene—Trl _en82 . The mutation is the insertion of a 1.4 kb P-element into the 5′ untranslated region. Trl expression decreased in the ovaries of mutant flies by about 30%; however, it caused abnormalities. The Trl _en82 mutation combined with the null allele of Trl caused female sterility: the females laid a few small eggs with abnormal shape. Many egg chambers demonstrated abnormalities in the Trl _en82 mutants: the oocyte had a regular shape and intruded into the egg chamber region with nurse cells; the rapid transport of nurse cell cytoplasm into the oocyte was disturbed, which resulted in the “dumpless” phenotype of the chambers in mutants; follicular cells often did not completely cover the oocyte and concentrated on its posterior end; and the migration of centripetal cells was affected. We propose that the sterility of the Trl _en82 females is due to the abnormal functioning of follicular cells resulting from low Trlexpression. This proposal is confirmed by normalizing the mutant phenotype of Trl _en82 females after the transfection of Trl cDNA. Note that even an insignificant decrease in Trl expression in such females seriously affected the somatic cell functioning, while a significant decrease in its expression in strong hypomorphic mutants affected both somatic and germline cells in the egg chambers.     

Attenuation of epigenetic regulator SMARCA4 and ERK‐ETS signaling suppresses aging‐related dopaminergic degeneration

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co‐expression analysis on human patient substantia nigra‐specific microarray datasets to identify potential novel disease‐related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin‐remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging‐dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down‐regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α‐synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK‐ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down‐regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD‐associated gene)‐deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age‐related disorders including PD.     

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.     

New phenotypes associated with the swallow gene of Drosophila: evidence for a general role in oocyte cytoskeletal organization

During oogenesis in Drosophila, several mRNAs and proteins are localized to discrete regions of the developing oocyte, resulting in a mature oocyte with a well-defined anterior–posterior axis. The product of the swallow (sww) gene is required for the localization of two different mRNAs during oogenesis, bicoid (bcd) and Adducin-like/hu-li tai shao (hts). We initiated a detailed characterization of the phenotypes associated with each of eight sww alleles as a means of investigating the role of sww in oogenic patterning. RNA localization defects in various sww mutants were examined by radioactive in situ hybridization to paraffin sections. Using this technique, several previously unreported RNA localization defects have been observed. Although bcd RNA localization is often lost completely in sww oocytes, in a high proportion of cases, bcd RNA is localized inappropriately along the periphery of the mature oocyte. In several sww mutants, a portion of the bcd mRNA population becomes concentrated at the posterior pole of the oocyte during late oogenesis. Several sww mutations also result inoskar RNA localization defects, consistent with a global role for sww in cytoskeletal regulation or organization. A detailed temporal and spatial analysis of hts RNA localization in sww mutants and in drug-treated ovaries reveals many similarities to bcd RNA localization, and implies the two independent localization events are accomplished by the same mechanism. Received: 10 January 2000 / Accepted: 9 March 2000     

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL‐1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro. We detect endogenous linear ubiquitin chain‐derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination‐defective flies by mutating residues essential for the catalytic activity of LUBEL. Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.     

A genomic investigation of ecological differentiation between free‐living and Drosophila‐associated bacteria

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome‐scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.     

Neuronal glycogen synthesis contributes to physiological aging

Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss‐of‐function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide‐based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild‐type laboratory mice also accumulate glycogen‐based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress‐response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age‐related neurological decline in humans.     

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, we analyzed BMP signaling in the FCs of 16 Drosophila species that span 45 million years of evolution. We found that the spatial patterns of BMP signaling in the FCs are dynamic and exhibit a range of interspecies' variations. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies' variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. Our results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning.     

Mutations in the gene stand still disrupt germ cell differentiation in Drosophila ovaries

During oogenesis in Drosophila, germ cells appear in sequential clusters of 16 interconnected cells. The events surrounding the differentiation of these cells are not fully understood. Here we present genetic and morphological analysis of mutations in the gene stand still (stil). Through complementation analyses we have refined the location of this gene to cyological region 49B-C. Our analyses of ovaries from ethylmethane sulfonate (EMS) - induced mutant alleles of this gene suggest that mutations in the stil gene produce a wide range of phenotypic abnormalities, from the absence of germ cells in the most severe alleles, to egg chambers with cytoskeletal defects in the less severe alleles. Our results suggest a role for this gene in specifying or maintaining a cytoskeletal component, with consequences during oogenesis and possibly during germ line sex determination. © 1996 Wiley-Liss, Inc.