Review Article

Demontis F, Piccirillo R, Goldberg AL, Perrimon N. The influence of skeletal muscle on systemic aging and lifespan. Aging Cell. 2013;12 (6) :943-9. Abstract

Epidemiological studies in humans suggest that skeletal muscle aging is a risk factor for the development of several age-related diseases such as metabolic syndrome, cancer, Alzheimer's and Parkinson's disease. Here, we review recent studies in mammals and Drosophila highlighting how nutrient- and stress-sensing in skeletal muscle can influence lifespan and overall aging of the organism. In addition to exercise and indirect effects of muscle metabolism, growing evidence suggests that muscle-derived growth factors and cytokines, known as myokines, modulate systemic physiology. Myokines may influence the progression of age-related diseases and contribute to the intertissue communication that underlies systemic aging.

2013_Aging Cell_Demontis.pdf
Mohr SE, Perrimon N. RNAi screening: new approaches, understandings, and organisms. Wiley Interdiscip Rev RNA. 2012;3 (2) :145-58. Abstract

RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity, and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.

Perrimon N, Zirin J, Bai J. Primary cell cultures from Drosophila gastrula embryos. J Vis Exp. 2011;(48). Abstract

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

2011_J Vis Exp_Perrimon.pdf
Rajan A, Perrimon N. Drosophila as a model for interorgan communication: lessons from studies on energy homeostasis. Dev Cell. 2011;21 (1) :29-31. Abstract

Current studies of physiological communication between Drosophila organs are beginning to address the fundamental problem of how nutrients regulate organismal growth, stem cell behavior, immunity, and aging. Advances in the Drosophila genetic tool kit will allow the design of genetic screens to systematically identify factors involved in organ communication.

2011_Dev Cell_Rajan.pdf
Perrimon N, Barkai N. The era of systems developmental biology. Curr Opin Genet Dev. 2011;21 (6) :681-3. 2011_Curr Op Gen Dev_Perrimon.pdf
Neumüller RA, Perrimon N. Where gene discovery turns into systems biology: genome-scale RNAi screens in Drosophila. Wiley Interdiscip Rev Syst Biol Med. 2011;3 (4) :471-8. Abstract

Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline. WIREs Syst Biol Med 2011 3 471-478 DOI: 10.1002/wsbm.127

Karpowicz P, Perrimon N. All for one, and one for all: the clonality of the intestinal stem cell niche. F1000 Biol Rep. 2010;2 :73. Abstract

Intestinal epithelia are maintained by intestinal stem cells (ISCs) that divide to replace dying absorptive and secretory cells that make up this tissue. Lineage labeling studies, both in vertebrates and Drosophila, have revealed the relationships between ISCs and their progeny. In addition, a number of signaling pathways involved in ISC proliferation and differentiation have been identified. Further studies will clarify the signals originating from the ISC niche and determine the processes that control the number and uniform distribution of niches throughout the epithelium.

Rajan A, Perrimon N. Steroids make you bigger? Fat chance says Myc. Cell Metab. 2010;12 (1) :7-9. Abstract

In flies, ecdysone integrates growth with developmental transitions by antagonizing insulin signaling, which links growth with nutritional status. Work in Developmental Cell (Delanoue et. al, 2010) finds that ecdysone represses the transcription factor Myc in the larval fat body to inhibit systemic growth, revealing a mechanism for such coordination.

2010_Cell Metab_Rajan.pdf
Zirin J, Perrimon N. Drosophila as a model system to study autophagy. Semin Immunopathol. 2010;32 (4) :363-72. Abstract

Originally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context.

2010_Semin Immunopathol_Zirin.pdf
Mohr S, Bakal C, Perrimon N. Genomic screening with RNAi: results and challenges. Annu Rev Biochem. 2010;79 :37-64. Abstract

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

2010_An Rev Biochem_Mohr.pdf
Bakal C, Perrimon N. Realizing the promise of RNAi high throughput screening. Dev Cell. 2010;18 (4) :506-7. Abstract

Recently reporting in Nature, Collinet et al. describes the application of quantitative multiparametric methods to a genome-wide RNAi screen for regulators of endocytosis. The study illustrates the power of this approach beyond the identification of new endocytic components to providing insights into the design principles of the endocytic system.

2010_Dev Cell_Bakal.pdf
Vinegoni C, Razansky D, Pitsouli C, Perrimon N, Ntziachristos V, Weissleder R. Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila. J Vis Exp. 2009;(30). Abstract

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size. We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Bai J, Sepp KJ, Perrimon N. Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. Nat Protoc. 2009;4 (10) :1502-12. Abstract

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.

2009_Nat Protocols_Bai.pdf
Pitsouli C, Apidianakis Y, Perrimon N. Homeostasis in infected epithelia: stem cells take the lead. Cell Host Microbe. 2009;6 (4) :301-7. Abstract

To maintain tissue homeostasis and avoid disease, epithelial cells damaged by pathogens need to be readily replenished, and this is mainly achieved by the activation of stem cells. In this Short Review, we discuss recent developments in the exciting field of host epithelia-pathogen interaction in Drosophila as well as in mammals.

2009_Cell Host_Pitsouli.pdf
Pitsouli C, Perrimon N. Developmental biology: Our fly cousins' gut. Nature. 2008;454 (7204) :592-3. 2008_Nature_Pitsouli.pdf
Perrimon N, Friedman A, Mathey-Prevot B, Eggert US. Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens. Drug Discov Today. 2007;12 (1-2) :28-33. Abstract

RNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.

2007_Drug Discov Today_Perrimon.pdf
Perrimon N, Mathey-Prevot B. Matter arising: off-targets and genome-scale RNAi screens in Drosophila. Fly (Austin). 2007;1 (1) :1-5. Abstract

Recently, the issue of off-target effects (OTEs) associated with long double stranded RNAs (dsRNAs) used in RNAi screens, such as those performed at the Drosophila RNAi Screening Center and other laboratories, has become a focus of great interest and some concern. Although OTEs have been recognized as an important source of false positives in mammalian studies (where short siRNAs are used as triggers), they were generally thought to be inconsequential in Drosophila RNAi experiments because of the use of long dsRNAs. Two recent papers have disputed this contention and show that significant off-target effects can take place with the use of some long dsRNAs in Drosophila cells. Together, these studies provide evidence that OTEs mediated by short homology stretches of 19nt or greater within long dsRNAs can contribute to false positives in Drosophila RNAi screens. Here, we address how widespread the occurrence of OTE is in Drosophila screens, focusing on the DRSC dsRNA collections, and we discuss the implication for the interpretation of results reported in RNAi screens to-date. Lastly, we summarize steps taken by the DRSC to redress that situation and include a set of recommendations to observe in future RNAi screens.

Ramadan N, Flockhart I, Booker M, Perrimon N, Mathey-Prevot B. Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat Protoc. 2007;2 (9) :2245-64. Abstract

This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.

2007_Nat Prot_Ramadan.pdf
Mathey-Prevot B, Perrimon N. Do-it-yourself RNAi made easy?. Nat Methods. 2007;4 (4) :308-9. 2007_Nat Meth_Mathey-Prevot.pdf
Leopold P, Perrimon N. Drosophila and the genetics of the internal milieu. Nature. 2007;450 (7167) :186-8. Abstract

'Homeostasis', from the Greek words for 'same' and 'steady', refers to ways in which the body acts to maintain a stable internal environment despite perturbations. Recent studies in Drosophila exemplify the conservation of regulatory mechanisms involved in metabolic homeostasis. These new findings underscore the use of Drosophila as a model for the study of various human disorders.