iPLA2-VIA is required for healthy aging of neurons, muscle, and the female germline in Drosophila melanogaster.

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

Using Drosophila melanogaster To Discover Human Disease Genes: An Educational Primer for Use with "Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target".

Since the dawn of the 20th century, the fruit fly Drosophila melanogaster has been used as a model organism to understand the nature of genes and how they control development, behavior, and physiology. One of the most powerful experimental approaches employed in Drosophila is the forward genetic screen. In the 21st century, genome-wide screens have become popular tools for identifying evolutionarily conserved genes involved in complex human diseases. In the accompanying article "Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal thePhospholipase DPathway as a Potential Therapeutic Target," Kankel and colleagues describe a forward genetic modifier screen to discover factors that contribute to the severe neurodegenerative disease amyotrophic lateral sclerosis (ALS). This primer briefly traces the history of genetic screens in Drosophila and introduces students to ALS. We then provide a set of guided reading questions to help students work through the data presented in the research article. Finally, several ideas for literature-based research projects are offered as opportunities for students to expand their appreciation of the potential scope of genetic screens. The primer is intended to help students and instructors thoroughly examine a current study that uses forward genetics in Drosophila to identify human disease genes.

Male reproductive aging arises via multifaceted mating-dependent sperm and seminal proteome declines, but is postponable in Drosophila.

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.

A course-based undergraduate research experience examining neurodegeneration in Drosophila melanogaster teaches students to think, communicate, and perform like scientists.

As educators strive to incorporate more active learning and inquiry-driven exercises into STEM curricula, Course-based Undergraduate Research Experiences (CUREs) are becoming more common in undergraduate laboratory courses. Here we detail a CURE developed in an upper-level undergraduate genetics course at Yeshiva University, centered on the Drosophila melanogaster ortholog of the human neurodegeneration locus PLA2G6/PARK14. Drosophila PLA2G6 mutants exhibit symptoms of neurodegeneration, such as attenuated lifespan and decreased climbing ability with age, which can be replicated by neuron-specific knockdown of PLA2G6. To ask whether the neurodegeneration phenotype could be caused by loss of PLA2G6 in specific neuronal subtypes, students used GAL4-UAS to perform RNAi knockdown of PLA2G6 in subsets of neurons in the Drosophila central nervous system and measured age-dependent climbing ability. We organized our learning objectives for the CURE into three broad goals of having students think, communicate, and perform like scientists. To assess how well students achieved these goals, we developed a detailed rubric to analyze written lab reports, administered pre- and post-course surveys, and solicited written feedback. We observed striking gains related to all three learning goals, and students reported a high degree of satisfaction. We also observed significantly improved understanding of the scientific method by students in the CURE as compared to the prior year's non-CURE genetics lab students. Thus, this CURE can serve as a template to successfully engage students in novel research, improve understanding of the scientific process, and expose students to the use of Drosophila as a model for human neurodegenerative disease.

Combover interacts with the axonemal component Rsp3 and is required for Drosophila sperm individualization.

Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. Here, we show that Combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes, and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes.

New genes often acquire male-specific functions but rarely become essential in Drosophila.

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.

Co-culture Activation of MAP Kinase in Drosophila S2 Cells.

Intercellular communication often involves phosphorylation of signal transduction proteins, including mitogen-activated protein kinases (MAPKs). Immunological detection of phosphorylated MAPK can be used to monitor signaling in vivo, identify novel pathway components, and assess ligand activity. In this chapter, I describe a cell co-culture method to assess activity of cell-bound extracellular ligands that result in phosphorylation of the ERK (extracellular signal-regulated kinase) MAPK in Drosophila. This protocol may be adaptable to other pathways and/or model systems.

Drosophila spermatid individualization is sensitive to temperature and fatty acid metabolism.

Fatty acids are precursors of potent lipid signaling molecules. They are stored in membrane phospholipids and released by phospholipase A2 (PLA2). Lysophospholipid acyltransferases (ATs) oppose PLA2 by re-esterifying fatty acids into phospholipids, in a biochemical pathway known as the Lands Cycle. Drosophila Lands Cycle ATs oys and nes, as well as 7 predicted PLA2 genes, are expressed in the male reproductive tract. Oys and Nes are required for spermatid individualization. Individualization, which occurs after terminal differentiation, invests each spermatid in its own plasma membrane and removes the bulk of the cytoplasmic contents. We developed a quantitative assay to measure individualization defects. We demonstrate that individualization is sensitive to temperature and age but not to diet. Mutation of the cyclooxygenase Pxt, which metabolizes fatty acids to prostaglandins, also leads to individualization defects. In contrast, modulating phospholipid levels by mutation of the phosphatidylcholine lipase Swiss cheese (Sws) or the ethanolamine kinase Easily shocked (Eas) does not perturb individualization, nor does Sws overexpression. Our results suggest that fatty acid derived signals such as prostaglandins, whose abundance is regulated by the Lands Cycle, are important regulators of spermatogenesis.

Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues.

Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

A screen for X-linked mutations affecting Drosophila photoreceptor differentiation identifies Casein kinase 1α as an essential negative regulator of wingless signaling.

The Wnt and Hedgehog signaling pathways are essential for normal development and are misregulated in cancer. The casein kinase family of serine/threonine kinases regulates both pathways at multiple levels. However, it has been difficult to determine whether individual members of this family have distinct functions in vivo, due to their overlapping substrate specificities. In Drosophila melanogaster, photoreceptor differentiation is induced by Hedgehog and inhibited by Wingless, providing a sensitive system in which to identify regulators of each pathway. We used a mosaic genetic screen in the Drosophila eye to identify mutations in genes on the X chromosome required for signal transduction. We recovered mutations affecting the transcriptional regulator CREB binding protein, the small GTPase dynamin, the cytoskeletal regulator Actin-related protein 2, and the protein kinase Casein kinase 1α. Consistent with its reported function in the ß-Catenin degradation complex, Casein Kinase 1α mutant cells accumulate ß-Catenin and ectopically induce Wingless target genes. In contrast to previous studies based on RNA interference, we could not detect any effect of the same Casein Kinase 1α mutation on Hedgehog signaling. We thus propose that Casein kinase 1α is essential to allow ß-Catenin degradation and prevent inappropriate Wingless signaling, but its effects on the Hedgehog pathway are redundant with other Casein kinase 1 family members.

Drosophila lysophospholipid acyltransferases are specifically required for germ cell development.

Enzymes of the membrane-bound O-acyltransferase (MBOAT) family add fatty acyl chains to a diverse range of protein and lipid substrates. A chromosomal translocation disrupting human MBOAT1 results in a novel syndrome characterized by male sterility and brachydactyly. We have found that the Drosophila homologues of MBOAT1, Oysgedart (Oys), Nessy (Nes), and Farjavit (Frj), are lysophospholipid acyltransferases. When expressed in yeast, these MBOATs esterify specific lysophospholipids preferentially with unsaturated fatty acids. Generating null mutations for each gene allowed us to identify redundant functions for Oys and Nes in two distinct aspects of Drosophila germ cell development. Embryos lacking both oys and nes show defects in the ability of germ cells to migrate into the mesoderm, a process guided by lipid signals. In addition, oys nes double mutant adult males are sterile due to specific defects in spermatid individualization. oys nes mutant testes, as well as single, double, and triple mutant whole adult animals, show an increase in the saturated fatty acid content of several phospholipid species. Our findings suggest that lysophospholipid acyltransferase activity is essential for germline development and could provide a mechanistic explanation for the etiology of the human MBOAT1 mutation.

Lipid-modified morphogens: functions of fats.

Despite their location in the aqueous extracellular environment, a number of secreted proteins carry hydrophobic lipid modifications. These modifications include glycosylphosphatidylinositol, cholesterol, and both saturated and unsaturated fatty acids, and they are attached in the secretory pathway by different classes of enzymes. Lipid attachments make crucial contributions to protein function in vivo through a diverse array of mechanisms. They can promote protein maturation and secretion, membrane tethering, targeting to specific membrane subdomains, or receptor binding and activation. Additionally, secretion of lipid-modified morphogens of the Wnt and Hh families requires dedicated accessory proteins and may involve their packaging into lipoprotein particles for long-range transport.

Microtubule polarity and axis formation in the Drosophila oocyte.

The body axes of the fruit fly are established in mid-oogenesis by the localization of three mRNA determinants, bicoid, oskar, and gurken, within the oocyte. General mechanisms of RNA localization and cell polarization, applicable to many cell types, have emerged from investigation of these determinants in Drosophila oogenesis. Localization of these RNAs is dependent on the germline microtubules, which reorganize to form a polarized array at mid-oogenesis in response to a signaling relay between the oocyte and the surrounding somatic follicle cells. Here we describe what is known about this microtubule reorganization and the signaling relay that triggers it. Recent studies have identified a number of ubiquitous RNA binding proteins essential for this process. So far, no targets for any of these proteins have been identified, and future work will be needed to illuminate how they function to reorganize microtubes and whether similar mechanisms also exist in other cell types.

The RNA-binding protein Squid is required for the establishment of anteroposterior polarity in the Drosophila oocyte.

The heterogeneous nuclear ribonucleoprotein (hnRNP) Squid (Sqd) is a highly abundant protein that is expected to bind most cellular RNAs. Nonetheless, Sqd plays a very specific developmental role in dorsoventral (DV) axis formation during Drosophila oogenesis by localizing gurken (grk) RNA. Here, we report that Sqd is also essential for anteroposterior (AP) axis formation. We identified sqd in a screen for modifiers of the Protein Kinase A (PKA) oogenesis polarity phenotype. The AP defects of sqd mutant oocytes resemble those of PKA mutants in several ways. In both cases, the cytoskeletal reorganization at mid-oogenesis, which depends on a signal from the posterior follicle cells, does not produce a correctly polarized microtubule (MT) network. This causes the posterior determinant, oskar (osk) RNA, to localize to central regions of the oocyte, where it is ectopically translated. Additionally, MT-dependent anterior movement of the oocyte nucleus and the grk-dependent specification of posterior follicle cells are unaffected in both mutants. However, in contrast to PKA mutants, sqd mutants do not retain a discrete posterior MT organizing center (MTOC) capable of supporting ectopic posterior localization of bicoid (bcd) RNA. sqd mutants also display several other phenotypes not seen in PKA mutants; these probably result from the disruption of MT polarity in earlier stages of oogenesis. Loss of Sqd does not affect polarity in follicle cells, wings or eyes, indicating a specific role in the determination of MT polarity within the germline.