Search Results (25)

We identified Murashka, a RING finger protein, in an oogenesis screen as a regulator of Drosophila ovary germline stem cell niche development. Mutant alleles of murashka exhibited an enlarged niche phenotype reminiscent of increased Notch signalling and displayed genetic interactions with Notch alleles, and with polychaetoid, a regulator of Notch during niche development. These interactions uncovered both positive and negative impacts on Notch in different genetic backgrounds. In S2 cells, Murashka formed a complex with Notch and colocalised with Notch in the secretory pathway. Murashka expression in S2 cells down-regulated Notch signalling levels but could result in increased fold induction due to the proportionally greater decrease in basal ligand-independent activity. In vivo Murashka expression had different outcomes on different Notch target genes. We observed a decrease in the expression of vestigial along the anterior/posterior boundary of the wing imaginal disc, but not of wingless at the dorsal/ventral boundary. Instead, weak ectopic wingless was observed, which was synergistically increased by the coexpression of Deltex, a positive regulator of ligand-independent signalling. Our results identify a novel developmental role for murashka, a gene previously only associated with a function in long-term memory, and indicate a regulatory role for Murashka through a physical interaction with Notch that has context-dependent outcomes. Murashka adds to a growing number of ubiquitin ligase regulators which interact with Notch at different locations within its secretory and endocytic trafficking pathways. Full article

In addition to energy storage, adipose tissue communication to other organs plays a key role in regulating organismal physiology. While the link between adipose tissue dysfunction and pathophysiology, including diabetes, chronic inflammation, and infertility, is clear, the molecular mechanisms that underlie these associations have not been fully described. We use Drosophila melanogaster as a model to better understand how adipose tissue communicates to the ovary. In this study, we utilized D. melanogaster’s robust genetic toolkit to examine the role of five adipokines known to control larval growth during development, CCHamide-1, CCHamide-2, eiger, Growth-blocking peptide 3, and unpaired 2 in regulating oogenesis. We show that the adult fat body expresses these “larval” adipokines. Our data indicate that ovarian germline stem cell maintenance does not require these adipokines. However, adipocyte-derived CCHamide-1, eiger, Growth-blocking peptide 3, and unpaired 2 influence early and late germline survival as well as ovulation. Thus, this work uncovers several adipokines that mediate fat-to-ovary communication. Full article

The fruit fly, Drosophila melanogaster, is an experimentally tractable model system that has recently emerged as a powerful “new approach methodology” (NAM) for chemical safety testing. As oogenesis is well conserved at the molecular and cellular level, measurements of Drosophila fecundity can be useful for identifying chemicals that affect reproductive health across species. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another, the RoboCam, a low-cost, custom-built robotic camera to capture images of the wells automatically, and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r² = 0.98 for the correlation between the automated egg counts and the ground truth). In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high-throughput egg-laying assays that require exposing flies to multiple different media conditions. Full article

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDC^mel complex in hybrid ovaries was not assembled. However, the activity of the RDC^sim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasa^sim allele and a significant zygotic delay in vasa^mel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species. Full article

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)^SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)^sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions. Full article

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses. Full article

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live-cell imaging to capture the continuous dynamics of cytoophidia in Stages 10–12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis. Full article

Oogenesis is a developmental process leading to the formation of an oocyte, a haploid gamete, which upon fertilisation and sperm entry allows the male and the female pronuclei to fuse and give rise to a zygote. In addition to forming a haploid gamete, oogenesis builds up a store of proteins, mRNAs, and organelles in the oocyte needed for the development of the future embryo. In several species, such as Drosophila, the polarity axes determinants of the future embryo must be asymmetrically distributed prior to fertilisation. In the Drosophila oocyte, the correct positioning of the nucleus is essential for establishing the dorsoventral polarity axis of the future embryo and allowing the meiotic spindles to be positioned in close vicinity to the unique sperm entry point into the oocyte. Full article

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOsp^F mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular. Full article

We investigated the effects of dietary delivered self-DNA in the model insect Drosophila melanogaster. Self-DNA administration resulted in low but significant lethality in Drosophila larvae and considerably extended the fly developmental time. This was characterized by the abnormal persistence of the larvae in the L2 and L3 stages, which largely accounted for the average 72 h delay observed in pupariation, as compared to controls. In addition, self-DNA exposure affected adult reproduction by markedly reducing both female fecundity and fertility, further demonstrating its impact on Drosophila developmental processes. The effects on the metabolites of D. melanogaster larvae after exposure to self-DNA were studied by NMR, LC-MS, and molecular networking. The results showed that self-DNA feeding reduces the amounts of all metabolites, particularly amino acids and N-acyl amino acids, which are known to act as lipid signal mediators. An increasing amount of phloroglucinol was found after self-DNA exposure and correlated to developmental delay and egg-laying suppression. Pidolate, a known intermediate in the γ-glutamyl cycle, also increased after exposure to self-DNA and correlated to the block of insect oogenesis. Full article

The eggshell that surrounds insect eggs acts as a barrier, protecting against biotic factors and desiccation. The eggshell is a multi-layered structure which is synthesised by the somatic follicle cells that surround the developing oocyte. Although the temporal order of expression of the protein eggshell components goes someway to explaining how the different layers are built up, but how the precise three-dimensional structure is achieved and how lipid components responsible for desiccation resistance are incorporated are poorly understood. In this paper, we demonstrate that wunen, which encodes a lipid phosphate phosphatase, is necessary for fertility in Drosophila females. Compared to sibling controls, females null for wunen lay fewer eggs which subsequently collapse such that no larvae emerge. We show that this is due to a requirement for wunen in the ovarian follicle cells which is needed to produce an ordered and functional eggshell. Knockdown of a septate junction component also results in collapsed eggs, supporting the idea that similar to its role in embryonic tracheal development, Wunen in follicle cells also promotes septate junction function. Full article

Primary oocyte determination occurs in many organisms within a germ line cyst, a multicellular structure composed of interconnected germ cells. However, the structure of the cyst is itself highly diverse, which raises intriguing questions about the benefits of this stereotypical multicellular environment for female gametogenesis. Drosophila melanogaster is a well-studied model for female gametogenesis, and numerous genes and pathways critical for the determination and differentiation of a viable female gamete have been identified. This review provides an up-to-date overview of Drosophila oocyte determination, with a particular emphasis on the mechanisms that regulate germ line gene expression. Full article

Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered. Full article

RNA binding proteins (RBPs) play a fundamental role in the post-transcriptional regulation of gene expression within the germline and nervous system. This is underscored by the prevalence of mutations within RBP-encoding genes being implicated in infertility and neurological disease. We previously described roles for the highly conserved RBP Caper in neurite morphogenesis in the Drosophila larval peripheral system and in locomotor behavior. However, caper function has not been investigated outside the nervous system, although it is widely expressed in many different tissue types during embryogenesis. Here, we describe novel roles for Caper in fertility and mating behavior. We find that Caper is expressed in ovarian follicles throughout oogenesis but is dispensable for proper patterning of the egg chamber. Additionally, reduced caper function, through either a genetic lesion or RNA interference-mediated knockdown of caper in the female germline, results in females laying significantly fewer eggs than their control counterparts. Moreover, this phenotype is exacerbated with age. caper dysfunction also results in partial embryonic and larval lethality. Given that caper is highly conserved across metazoa, these findings may also be relevant to vertebrates. Full article

We perform quantitative studies to investigate the effect of high-calorie diet on Drosophila oogenesis. We use the central composite design (CCD) method to obtain quadratic regression models of body fat and fertility as a function of the concentrations of protein and sucrose, two major macronutrients in Drosophila diet, and treatment duration. Our results reveal complex interactions between sucrose and protein in impacting body fat and fertility when they are considered as an integrated physiological response. We verify the utility of our quantitative modeling approach by experimentally confirming the physiological responses—including increased body fat, reduced fertility, and ovarian insulin insensitivity—expected of a treatment condition identified by our modeling method. Under this treatment condition, we uncover a Drosophila oogenesis phenotype that exhibits an accumulation of immature oocytes and a halt in the production of mature oocytes, a phenotype that bears resemblance to key aspects of the human condition of polycystic ovary syndrome (PCOS). Our analysis of the dynamic progression of different aspects of diet-induced pathophysiology also suggests an order of the onset timing for obesity, ovarian dysfunction, and insulin resistance. Thus, our study documents the utility of quantitative modeling approaches toward understanding the biology of Drosophila female reproduction, in relation to diet-induced obesity and type II diabetes, serving as a potential disease model for human ovarian dysfunction. Full article