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2021
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.
2021_TiG_Zirin.pdf
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.
2021_SciAdv_Chatterjee.pdf
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.
2021_COIS_Hung.pdf
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.
2021_WileyDevBio_Bosch.pdf
2020
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.
2020_AgeResRev_Parkhitko.pdf
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.
2020_CurrProt_Bosch.pdf
2019
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.
2019_DMM_Mohr.pdf
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.
2019_WIRES_Ahmad.pdf
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
2018
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.
2018_BioEssays_Song.pdf
Xu C, Ericsson M, Perrimon N. Understanding cellular signaling and systems biology with precision: A perspective from ultrastructure and organelle studies in the Drosophila midgut. Current Opinion in Systems Biology [Internet]. 2018;(11) :24-31. Publisher's VersionAbstract

The adult Drosophila midgut is a complex tissue with various cell types that interact closely to maintain tissue integrity and perform organ function. The gut consists of a pseudostratified epithelium, a latticework of circular and longitudinal visceral muscles that supports the epithelium, and a tracheal vascular system. The major cell types of the midgut epithelium are the absorptive enterocytes (ECs), characterized by a large nucleus and microvilli-covered luminal surface, the enteroendocrine cells (EEs) that produce various hormones, and the intestinal stem cells (ISCs) that produce ECs and EEs [1,2] . Interactions between these cell types are critical to maintaining tissue integrity and gut function. For example, ISCs proliferation and differentiation are controlled by a complex network integrating autocrine and paracrine signals [3,4] ; hormones derived from EEs regulate EC physiology; and EC-derived factors signal to ISCs following gut damage.

 

2018_Curr Op Sys Bio_Xu.pdf
2017
Housden BE, Nicholson HE, Perrimon N. Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Cells. Bio Protoc. 2017;7 (3). Abstract
A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In , combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden , 2015).
2017_Bio Protoc_Housden.pdf
Ewen-Campen B, Mohr SE, Hu Y, Perrimon N. Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR. Dev Cell. 2017;43 (1) :6-9. Abstract
Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.
2017_Dev Cell_Ewen-Campen.pdf
Chen C-L, Perrimon N. Proximity-dependent labeling methods for proteomic profiling in living cells. Wiley Interdiscip Rev Dev Biol. 2017;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 with biotin. The biotinylated 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 describe and compare current methods of proximity labeling as well as their applications. 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. For further resources related to this article, please visit the WIREs website.

2017_Interdis Rev Dev Bio_Chen.pdf
Chatterjee N, Perrimon N. Thermogenesis by THADA. Dev Cell. 2017;41 (1) :1-2. Abstract

THADA has been associated with cold adaptation and diabetes in humans, but the cellular and molecular basis of its function has been unknown. Moraru and colleagues (2017) report in this issue of Developmental Cell that it triggers thermogenesis by uncoupling ATP hydrolysis from calcium transport into the endoplasmic reticulum.

2017_Dev Cell_Chatterjee.pdf
Housden BE, Nicholson HE, Perrimon N. Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells. Bio-Protocol [Internet]. 2017;7 (3). Publisher's VersionAbstract

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

2017_Bio-Protocol_Housden.pdf
Housden BE, Muhar M, Gemberling M, Gersbach CA, Stainier DYR, Seydoux G, et al. Loss-of-function genetic tools for animal models: cross-species and cross-platform differences. Nat Rev Genet. 2017;18 (1) :24-40. Abstract
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
2016_Nat Rev Gene_Housden.pdf
2016
Droujinine IA, Perrimon N. Interorgan Communication Pathways in Physiology: Focus on Drosophila. Annu Rev Genet. 2016;Abstract

Studies in mammals and Drosophila have demonstrated the existence and significance of secreted factors involved in communication between distal organs. In this review, primarily focusing on Drosophila, we examine the known interorgan communication factors and their functions, physiological inducers, and integration in regulating physiology. Moreover, we describe how organ-sensing screens in Drosophila can systematically identify novel conserved interorgan communication factors. Finally, we discuss how interorgan communication enabled and evolved as a result of specialization of organs. Together, we anticipate that future studies will establish a model for metazoan interorgan communication network (ICN) and how it is deregulated in disease. Expected final online publication date for the Annual Review of Genetics Volume 50 is November 23, 2016. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

2016_Annu Rev Genet_Drouijinine.pdf

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