Review Article

Petsakou A, Perrimon N. Bioelectric regulation of intestinal stem cells. Trends in Cell Biology. 2022;Nov 14;S0962-8924 (22) :234-3. Abstract

Proper regulation of ion balance across the intestinal epithelium is essential for physiological functions, while ion imbalance causes intestinal disorders with dire health consequences. Ion channels, pumps, and exchangers are vital for regulating ion movements (i.e., bioelectric currents) that control epithelial absorption and secretion. Recent in vivo studies used the Drosophila gut to identify conserved pathways that link regulators of Ca2+, Na+ and Cl- with intestinal stem cell (ISC) proliferation. These studies laid a foundation for using the Drosophila gut to identify conserved proliferative responses triggered by bioelectric regulators. Here, we review these studies, discuss their significance, as well as the advantages of using Drosophila to unravel conserved bioelectrically induced molecular pathways in the intestinal epithelium under physiological, pathophysiological, and regenerative conditions.

Keywords: Drosophila; bioelectric signaling; gut; intestinal stem cells; ion channels.


Petsakou A, Perrimon N. No sugar, just protein please - says the fly. Nat Metab. 2022; 2022_naturemetabolism_petsakou.pdf
Huang K, Liu Y, Perrimon N. Roles of Insect Oenocytes in Physiology and Their Relevance to Human Metabolic Diseases. Front. Insect Sci. [Internet]. 2022; Publisher's VersionAbstract

Oenocytes are large secretory cells present in the abdomen of insects known to synthesize very-long-chain fatty acids to produce hydrocarbons and pheromones that mediate courtship behavior in adult flies. In recent years, oenocytes have been implicated in the regulation of energy metabolism. These hepatocyte-like cells accumulate lipid droplets under starvation and can non-autonomously regulate tracheal waterproofing and adipocyte lipid composition. Here, we summarize evidence, mostly from Drosophila, establishing that oenocytes perform liver-like functions. We also compare the functional differences in oenocytes and the fat body, another lipid storage tissue which also performs liver-like functions. Lastly, we examine signaling pathways that regulate oenocyte metabolism derived from other metabolic tissues, as well as oenocyte-derived signals that regulate energy homeostasis.


Regulating energy utilization and storage is central to animal physiology and adaptation to environmental challenges. Under conditions of nutrition surplus, glucose is converted to fatty acids, which are then synthesized into triglycerides (TGs) and stored as lipid droplets. Excessive lipid stores can be detrimental and have been associated with various metabolic diseases, such as cardiovascular diseases (CVDs), non-alcoholic fatty liver disease (NAFLD), obesity and insulin resistance, making understanding lipid metabolism of great importance to human health.

The liver is the major detoxifying organ of the body and plays a central role in regulating the metabolism of carbohydrates, proteins and lipids. Moreover, the liver is the major site for glycogen storage and very low-density lipoprotein (VLDL) secretion (12). During starvation, adipocytes undergo lipolysis to produce free fatty acids (FFAs). FFAs are processed by hepatic oxidation to generate ketone bodies in the liver which are then used as fuels for other tissues. If mobilization of FFAs exceeds the rate of lipid oxidation, re-esterification of surplus FFAs to TGs occurs in the liver, leading to an increase in intrahepatic TG content, i.e., steatosis. NAFLD, a common manifestation of the metabolic syndrome, is characterized by steatosis in the absence of starvation. Nonalcoholic hepatic steatosis is present in approximately 25% of the adult population worldwide, and NAFLD is the most common liver disease in Western societies. Thus, understanding how hepatic diseases regulate cellular processes in peripheral organs and how other organs contribute to steatosis is of interest to human metabolic diseases.

Major metabolic and endocrine pathways are conserved in Drosophila, making this model organism well suited to dissect the cellular and molecular mechanisms underlying physiology (35). The fly fat body is equivalent to the vertebrate white adipose tissue (WAT), which stores excess fat as TGs. In addition, fly oenocytes, which are similar to hepatocyte cells, are important for mobilizing stored lipids from the fly fat body (6). Like mammals, flies convert excess carbohydrates into TGs through de novo lipogenesis (78). In addition, excess carbohydrates and amino acids can also be processed into UDP-glucose, which fuels glycogen synthesis (9). Regulation of energy storage in flies involves several signaling pathways, including insulin/insulin-like growth factor (IGF) signaling, which is similar to the insulin signaling in mammals (10). However, unlike mammals, there are eight different Drosophila insulin-like peptides (dILPs). Most of these modulate the IGF pathway through a single insulin receptor, InR (1011). Under nutrient-deprivation or energy demanding conditions, lipids are released from the fat body through increased lipolysis (12), and are further processed in oenocytes (613). Signaling that regulates catabolism of lipids and carbohydrates include adipokinetic hormone (Akh), which is similar to glucagon in mammals and ecdysone, which antagonizes insulin signaling (1415).

In this review, we explore the potential of Drosophila oenocytes as a model for hepatic diseases. We summarize the different roles of oenocytes and the fat body in regulating carbohydrate and lipid metabolism under normal or starved conditions. We also discuss the intricate interplay of oenocytes with other tissues, including the fat body and muscles, in shaping organismal lipid storage.

Liu Y, Saavedra P, Perrimon N. Cancer cachexia: lessons from Drosophila. Dis Model Mech [Internet]. 2022;15 (3). PMC Full TextAbstract
Cachexia, a wasting syndrome that is often associated with cancer, is one of the primary causes of death in cancer patients. Cancer cachexia occurs largely due to systemic metabolic alterations stimulated by tumors. Despite the prevalence of cachexia, our understanding of how tumors interact with host tissues and how they affect metabolism is limited. Among the challenges of studying tumor-host tissue crosstalk are the complexity of cancer itself and our insufficient knowledge of the factors that tumors release into the blood. Drosophila is emerging as a powerful model in which to identify tumor-derived factors that influence systemic metabolism and tissue wasting. Strikingly, studies that are characterizing factors derived from different fly tumor cachexia models are identifying both common and distinct cachectic molecules, suggesting that cachexia is more than one disease and that fly models can help identify these differences. Here, we review what has been learned from studies of tumor-induced organ wasting in Drosophila and discuss the open questions.
Zirin J, Bosch J, Viswanatha R, Mohr SE, Perrimon N. State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila. Trends Genet. 2021;Abstract
For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.
Tattikota SG, Perrimon N. Preparation of Drosophila Larval Blood Cells for Single-cell RNA Sequencing. Bio-Protocol. 2021;11 (16). 2021_Bio-Protocol_Tattikota.pdf
Cable J, Pourquié O, Wellen KE, Finley LWS, Aulehla A, Gould AP, et al. Metabolic decisions in development and disease-a Keystone Symposia report. Ann N Y Acad Sci. 2021;Abstract
There is an increasing appreciation for the role of metabolism in cell signaling and cell decision making. Precise metabolic control is essential in development, as evident by the disorders caused by mutations in metabolic enzymes. The metabolic profile of cells is often cell-type specific, changing as cells differentiate or during tumorigenesis. Recent evidence has shown that changes in metabolism are not merely a consequence of changes in cell state but that metabolites can serve to promote and/or inhibit these changes. Metabolites can link metabolic pathways with cell signaling pathways via several mechanisms, for example, by serving as substrates for protein post-translational modifications, by affecting enzyme activity via allosteric mechanisms, or by altering epigenetic markers. Unraveling the complex interactions governing metabolism, gene expression, and protein activity that ultimately govern a cell's fate will require new tools and interactions across disciplines. On March 24 and 25, 2021, experts in cell metabolism, developmental biology, and human disease met virtually for the Keystone eSymposium, "Metabolic Decisions in Development and Disease." The discussions explored how metabolites impact cellular and developmental decisions in a diverse range of model systems used to investigate normal development, developmental disorders, dietary effects, and cancer-mediated changes in metabolism.
Chatterjee N, Perrimon N. What fuels the fly: Energy metabolism in and its application to the study of obesity and diabetes. Sci Adv. 2021;7 (24). Abstract
The organs and metabolic pathways involved in energy metabolism, and the process of ATP production from nutrients, are comparable between humans and Drosophila melanogaster This level of conservation, together with the power of Drosophila genetics, makes the fly a very useful model system to study energy homeostasis. Here, we discuss the major organs involved in energy metabolism in Drosophila and how they metabolize different dietary nutrients to generate adenosine triphosphate. Energy metabolism in these organs is controlled by cell-intrinsic, paracrine, and endocrine signals that are similar between Drosophila and mammals. We describe how these signaling pathways are regulated by several physiological and environmental cues to accommodate tissue-, age-, and environment-specific differences in energy demand. Last, we discuss several genetic and diet-induced fly models of obesity and diabetes that can be leveraged to better understand the molecular basis of these metabolic diseases and thereby promote the development of novel therapies.
Mohr SE, Tattikota SG, Xu J, Zirin J, Hu Y, Perrimon N. Methods and tools for spatial mapping of single-cell RNAseq clusters in Drosophila. Genetics. 2021;Abstract
Single-cell RNA sequencing (scRNAseq) experiments provide a powerful means to identify clusters of cells that share common gene expression signatures. A major challenge in scRNAseq studies is to map the clusters to specific anatomical regions along the body and within tissues. Existing data, such as information obtained from large-scale in situ RNA hybridization studies, cell type specific transcriptomics, gene expression reporters, antibody stainings, and fluorescent tagged proteins, can help to map clusters to anatomy. However, in many cases, additional validation is needed to precisely map the spatial location of cells in clusters. Several approaches are available for spatial resolution in Drosophila, including mining of existing datasets, and use of existing or new tools for direct or indirect detection of RNA, or direct detection of proteins. Here, we review available resources and emerging technologies that will facilitate spatial mapping of scRNAseq clusters at high resolution in Drosophila. Importantly, we discuss the need, available approaches, and reagents for multiplexing gene expression detection in situ, as in most cases scRNAseq clusters are defined by the unique coexpression of sets of genes.
Hung R-J, Li JSS, Liu Y, Perrimon N. Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics. Curr Opin Insect Sci. 2021;47 :12-17. Abstract
The Drosophila midgut has emerged in recent years as a model system to study stem cell renewal and differentiation and tissue homeostasis. Histological, genetic and gene expression studies have provided a wealth of information on gut cell types, regionalization, genes and pathways involved in cell proliferation and differentiation, stem cell renewal, and responses to changes in environmental factors such as the microbiota and nutrients. Here, we review the contribution of single cell transcriptomic methods to our understanding of gut cell type diversity, lineage and behavior.
Bosch JA, Chen C-L, Perrimon N. Proximity-dependent labeling methods for proteomic profiling in living cells: An update. Wiley Interdiscip Rev Dev Biol. 2021;10 (1) :e392. Abstract
Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins. The tagged endogenous proteins can then be isolated for further analysis by MS. To analyze protein-protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we provide an updated description and comparison of proximity labeling methods, as well as their applications and improvements. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions. This article is categorized under: Technologies > Analysis of Proteins.
Amamoto R, Garcia MD, West ER, Choi J, Lapan SW, Lane EA, et al. Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue. Bio Protoc. 2020;10 (18) :e3749. Abstract
Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, midgut, and developing chick retina, suggesting that it is likely useful for most organisms.
Parkhitko AA, Filine E, Mohr SE, Moskalev A, Perrimon N. Targeting metabolic pathways for extension of lifespan and healthspan across multiple species. Ageing Res Rev. 2020;64 :101188. Abstract
Metabolism plays a significant role in the regulation of aging at different levels, and metabolic reprogramming represents a major driving force in aging. Metabolic reprogramming leads to impaired organismal fitness, an age-dependent increase in susceptibility to diseases, decreased ability to mount a stress response, and increased frailty. The complexity of age-dependent metabolic reprogramming comes from the multitude of levels on which metabolic changes can be connected to aging and regulation of lifespan. This is further complicated by the different metabolic requirements of various tissues, cross-organ communication via metabolite secretion, and direct effects of metabolites on epigenetic state and redox regulation; however, not all of these changes are causative to aging. Studies in yeast, flies, worms, and mice have played a crucial role in identifying mechanistic links between observed changes in various metabolic traits and their effects on lifespan. Here, we review how changes in the organismal and organ-specific metabolome are associated with aging and how targeting of any one of over a hundred different targets in specific metabolic pathways can extend lifespan. An important corollary is that restriction or supplementation of different metabolites can change activity of these metabolic pathways in ways that improve healthspan and extend lifespan in different organisms. Due to the high levels of conservation of metabolism in general, translating findings from model systems to human beings will allow for the development of effective strategies for human health- and lifespan extension.
Ewen-Campen B, Perrimon N. Expanding the horizons of genome editing in the fruit fly with Cas12a. Proc Natl Acad Sci U S A. 2020;117 (39) :24019-24021. 2020_PNAS_Ewen-Campen.pdf
Bosch JA, Knight S, Kanca O, Zirin J, Yang-Zhou D, Hu Y, et al. Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes. Curr Protoc Mol Biol. 2020;130 (1) :e112. Abstract
The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.
Mohr SE, Perrimon N. Drosophila melanogaster: a simple system for understanding complexity. Dis Model Mech. 2019;12 (10). Abstract
Understanding human gene function is fundamental to understanding and treating diseases. Research using the model organism benefits from a wealth of molecular genetic resources and information useful for efficient experimentation. Moreover, offers a balance as a relatively simple organism that nonetheless exhibits complex multicellular activities. Recent examples demonstrate the power and continued promise of research to further our understanding of conserved gene functions.
Parkhitko AA, Jouandin P, Mohr SE, Perrimon N. Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species. Aging Cell. 2019;:e13034. Abstract
Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S-adenosylmethionine, which, after transferring its methyl group, is converted to S-adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.
2019_Aging Cell_Parkhitko.pdf
Ahmad M, He L, Perrimon N. Regulation of insulin and adipokinetic hormone/glucagon production in flies. Wiley Interdiscip Rev Dev Biol. 2019;:e360. Abstract
Metabolic homeostasis is under strict regulation of humoral factors across various taxa. In particular, insulin and glucagon, referred to in Drosophila as Drosophila insulin-like peptides (DILPs) and adipokinetic hormone (AKH), respectively, are key hormones that regulate metabolism in most metazoa. While much is known about the regulation of DILPs, the mechanisms regulating AKH/glucagon production is still poorly understood. In this review, we describe the various factors that regulate the production of DILPs and AKH and emphasize the need for future studies to decipher how energy homeostasis is governed in Drosophila. This article is categorized under: Invertebrate Organogenesis > Flies Signaling Pathways > Global Signaling Mechanisms.
He L, Ahmad M, Perrimon N. Mechanosensitive channels and their functions in stem cell differentiation. Exp Cell Res. 2019;374 (2) :259-265. Abstract
Stem cells continuously perceive and respond to various environmental signals during development, tissue homeostasis, and pathological conditions. Mechanical force, one of the fundamental signals in the physical world, plays a vital role in the regulation of multiple functions of stem cells. The importance of cell adhesion to the extracellular matrix (ECM), cell-cell junctions, and a mechanoresponsive cell cytoskeleton has been under intensive study in the fields of stem cell biology and mechanobiology. However, the involvement of mechanosensitive (MS) ion channels in the mechanical regulation of stem cell activity has just begun to be realized. Here, we review the diversity and importance of mechanosensitive channels (MSCs), and discuss recently discovered functions of MSCs in stem cell regulation, especially in the determination of cell fate.
2019_Exp Cell Res_He.pdf
Song W, Ghosh AC, Cheng D, Perrimon N. Endocrine Regulation of Energy Balance by Drosophila TGF-β/Activins. Bioessays. 2018;Abstract
The Transforming growth factor beta (TGF-β) family of secreted proteins regulates a variety of key events in normal development and physiology. In mammals, this family, represented by 33 ligands, including TGF-β, activins, nodal, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs), regulate biological processes as diverse as cell proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair, and endocrine functions. In Drosophila, only 7 members of this family are present, with 4 TGF-β/BMP and 3 TGF-β/activin ligands. Studies in the fly have illustrated the role of TGF-β/BMP ligands during embryogenesis and organ patterning, while the TGF-β/activin ligands have been implicated in the control of wing growth and neuronal functions. In this review, we focus on the emerging roles of Drosophila TGF-β/activins in inter-organ communication via long-distance regulation, especially in systemic lipid and carbohydrate homeostasis, and discuss findings relevant to metabolic diseases in humans.