Research Summary
Norbert Perrimon is using functional genomic approaches to identify molecular mechanisms that link physiology, cell biology, and cell differentiation.
The central objective of developmental biology is to understand how organisms grow to adulthood. In the past 30 years, studies using genetically tractable model systems have led to a detailed understanding of the genetic mechanisms involved in the control of developmental events, as illustrated by our intimate knowledge of patterning and morphogenesis. The next big questions are how complex phenotypes arise in the context of the whole organism and how the programs regulating their development and function are influenced by genetic background and environment. For example, little is understood about how the simultaneous growth and differentiation of different tissues are coordinated and how the development of different cell types and tissues is integrated within an organ. Furthermore, many of the mechanisms by which growth factor–triggered signaling events intersect with cell metabolism, which is regulated by nutrients and hormones, remain to be identified.
We are using Drosophila as a model system to characterize the responses of specific cells to extracellular signals. Our previous work focused on the characterization of the signaling pathways that orchestrate embryonic patterning and morphogenesis. More recently, however, as we now have a rather good knowledge of these processes, we have become interested in studying (1) the mechanisms involved in the control of cell and tissue growth, and especially the roles of the insulin pathway in these processes, and (2) how signaling mechanisms are used in the context of homeostasis. 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. We are interested in two kinds of homeostatic regulation: (1) physiological homeostasis, which encompasses the mechanisms by which differentiated tissues, such as muscles, grow and maintain their mass during the aging process; and (2) tissue/regenerative homeostasis, which addresses the maintenance of tissue integrity by stem cell systems, as in the gut, which exhibits slow regeneration under normal conditions but accelerated regeneration when injured. We are studying these fundamental problems in Drosophila because the fly is one of the prime model systems for studying the basis of human diseases and has an unmatched arsenal of tools for both in vivo and in vitro functional genomic studies.
Our ongoing work can be subdivided into four categories. First, to facilitate functional genomic approaches in Drosophila, we develop, improve, and generate reagent resources to make the process of gene discovery and identification of gene function—both in vivo and in vitro—faster, easier, more reliable, and genome-wide. To maintain and build on the Drosophila community's tradition of sharing, which was pivotal in the establishment of Drosophila as one of the premier model systems, we make the methods and reagents that we develop immediately available to the community. Second, we apply these tools to tissue culture cells to elucidate the organization of the core cell circuitry networks involved in signaling. Our approach, based on genome-wide RNA interference (RNAi) screening and proteomic and computational analyses, is to identify the parts responsible for the reception and integration of the signals, organize them into pathways and networks, and then validate the findings in more complex, in vivo biological systems, i.e., muscles and gut stem cells. Third, as a model for physiological homeostasis, we study Drosophila muscles to identify the molecular mechanisms involved in their growth, maintenance, and aging. Fourth, as a model for tissue/regenerative homeostasis, we study the mechanisms that control the proliferation of Drosophila adult gut stem cells in both normal and injured conditions.
Functional Genomics
Genome-wide high-throughput screening. The availability of the Drosophila genome sequence in 2000 provided us with an unprecedented resource for functional genomic studies. To address the issue that 75 percent of the genome is not functionally annotated at this time, and to systematically analyze the functions of the ~15,000 known or predicted genes, we established a high-throughput screening platform to conduct RNAi screens in Drosophila tissue culture cells in the 384-well plate format. Next, we established at Harvard Medical School a Drosophila RNAi Screening Center (www.flyrnai.org) that is open to the scientific community for performing screens based on the technology and "know-how" that we developed. One measure of success of the center is that more than 110 screens have been conducted in the past six years.
Genome-wide RNAi screens allow us, once an appropriate cell-based assay has been established, to identify most genes involved in the process under investigation in a few weeks. The development of this platform has required the establishment of a screening infrastructure, the generation of screening reagents and the development of new protocols. To complement the RNAi functional screens with proteomics data sets, we recently established a mass spectrometry workflow that allows us to systematically build "miniproteomes" around core pathways.
Using these reagents and methods, we have performed a number of genome-wide RNAi screens to interrogate the structure of signaling pathways, the interaction between host cells and various viral and bacterial pathogens, cell morphology, and other cell biological questions. These studies have identified a number of new components involved in the biological processes under scrutiny.
RNAi screens generate a large amount of data that need to be "mined" with existing literature, proteomic data sets, and transcriptome analyses to generate robust hypotheses for subsequent in vivo validation. The implementation of data mining is difficult and severely hampered by a shortage of robust and fully integrated tools. For example, the identification of orthologs is the most important first step for meaningful interspecies cross-reference and can be particularly challenging. To facilitate the analysis of the results from RNAi screens, we are developing a number of data-mining tools.
Transgenic RNA. Because results from the tissue culture screens need to be followed up by in vivo validation, we have improved methods for transgenic RNAi. First, we developed a new vector system, the VALIUM series, to drive the expression of long double-stranded RNAs (dsRNAs) under UAS control at defined genomic locations, using the phage phiC31 integrase method; this system overcomes some of the variability issues observed with previous P-element–based random transgenesis methods. Second, as the expression of long dsRNAs in the female germline does not work, we developed a new method based on small hairpin micro RNAs (shmiRNAs)hat effectively works to generate knockdown of gene activities in both the germline and soma. We are using this approach to generate a large-scale library to target most Drosophila genes. These lines, which we are making available to the community as they are being produced, can be used to quickly validate results from genome-wide RNAi screens as well as to conduct more classic genetic screens or in vivo studies.
Cell Circuitry
A simple way to view cell signaling is that specific receptors activate core pathways that function in almost all cells and tissues of the animal. In addition to these core pathways, a number of tissue-specific upstream regulators and downstream effectors exist to specify the various biological outcomes. In support of this idea, the metabolic consequences of manipulating insulin signaling depend strongly on the identity of the tissue in which the manipulation takes place. In the context of larval muscles, for example, insulin drives endoreplication and protein translation, whereas in gut stem cells, it is required for cell proliferation.
To dissect signaling pathways, we use genome-wide RNAi and proteomics approaches in Drosophila cell lines to identify core pathway components and, to some extent, cell-type-specific components. We (1) use combinations of genome-wide RNAi and large-scale mass spectrometry to identify the components of signaling networks; (2) model circuit topology by measuring the effects of perturbing these components on the structure of the proteome, transcriptional outputs, and cellular phenotypes; and (3) identify points of intersections, or "cross-talk nodes," between signaling pathways. We then validate the information from these studies in two biological systems, muscles and gut stem cells.
A central theme of our studies is the insulin/TOR pathway, which functions in almost all cells and tissues of the animal. The insulin pathway regulates cell size and cell growth, as well as processes such as carbohydrate metabolism, lipid metabolism, and autophagy, and exemplifies how growth is a readout of cellular metabolism. We are particularly interested in identifying how insulin signaling and metabolism intersect with the MAPK, JAK/STAT, JNK, and Hippo pathways.
Physiological Homeostasis
We have initiated a number of studies on muscle growth during Drosophila larval development. During larval growth, individual muscles grow almost 100-fold in four days. This tremendous growth occurs in the absence of a change in the number of nuclei and is easily visualized by the addition of new sarcomeres to the preexisting myofibrils. This process relies on an increase in ploidy of the existing nuclei, protein synthesis, and mitochondriogenesis and is regulated by the TOR pathway, which links amino acid and growth factor levels with muscle cell growth. During the growth phase, new sarcomeres are added to the muscle edge. Starvation reverses this process, as some sarcomeres are degraded by proteolysis and the cytoplasm is consumed by autophagy. Myofibrillar protein degradation occurs primarily through the proteasome, while autophagy breaks down the rest of the cytoplasm and organelles. The dynamic regulation of muscle growth provides a paradigm to address how physiology influences cell biology and differentiation. In addition, analysis of muscle growth and homeostasis may shed light on muscle wasting, which is associated with lack of exercise and a number of human diseases and is somehow prevented in hibernating animals. We are addressing a number of questions with regard to muscle growth and its maintenance. Specifically, we are interested in (1) the roles of the insulin pathway in muscle growth, (2) the roles of microRNAs in insulin and muscle homeostasis, (3) the roles of autophagy in muscle mass maintenance, and (4) the molecular mechanisms of muscle aging. We are particularly interested in using assays related to physiological homeostasis to test hypotheses generated from the results of our cell circuitry studies.
Tissue or Regenerative Homeostasis
Under normal tissue homeostasis, committed stem cells slowly divide to replace differentiated cells. When many cells are lost as a result of injury, they are replaced expediently by an increase in the rate of stem cell division. As new cells are produced, the damaged tissue is regenerated, eventually returning to its correct size and to normal homeostasis. We are using stem cells present in the adult Drosophila midgut system (intestinal stem cells, or ISCs) to identify the signaling pathways involved in stem cell proliferation and differentiation and to characterize how these processes are regulated in the context of injury.
The lineage of ISCs is simple, as these cells divide to produce enteroblasts that differentiate directly into enterocytes or enteroendocrine cells without further division. In the past few years, the Wnt/Wingless, insulin receptor, EGFR, JAK/STAT, and Notch pathways have been implicated in the division and differentiation of ISCs. In the case of injury, JNK signaling is activated in dying enterocytes, which secrete the Unpaired cytokines to activate JAK/STAT signaling in ISCs. In response, ISCs increase their rate of division. This regenerative response of ISCs ceases once the progeny of ISCs have replaced the cells lost due to injury. Most recently, we have demonstrated that the Hippo pathway, known for its function in the control of organ size, normally represses the activity of the Yorkie transcriptional coactivator in ISCs. Following injury, Hippo repression ceases and Yorkie activates downstream targets that drive ISC proliferation.
We are addressing a number of questions with regard to ISCs. Specifically, we are interested in (1) establishing cancer-like stem cell models by activating a number of oncogenes or down-regulating the activity of a number of tumor suppressors in the ISCs; (2) performing genetic and chemical whole-animal screens to identify genes and small molecules that are required for growth and survival of "cancer" stem cells, but not their wild-type counterparts; and (3) studying the effects of dietary restriction on homeostasis of the gut. Because many signaling pathways are activated in ISCs, we are interested in using the ISC cell as a system to validate the points of intersection, or cross-talk nodes, between the PI3K, MAPK, JNK, Hippo, and JAK/STAT pathways identified and/or predicted from our cell circuitry projects.
This work is also partially supported by grants from the National Institute of General Medical Sciences, the National Cancer Institute, and the National Institute of Diabetes and Digestive and Kidney Diseases.
As of May 30, 2012
