Viewpoint: the Future of Model Organisms in Human Disease Research

PERSPECTIVES

98. Plass, C. et al. Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS‑M. VIEWPOINT Nature Genet. 14, 106–109 (1996). 99. O’Neill, M. J., Ingram, R. S., Vrana, P. B. & Tilghman, S. M. Allelic expression of IGF2 in marsupials and birds. Dev. Genes Evol. 210, The future of model organisms in 18–20 (2000). 100. Rapkins, R. W. et al. Recent assembly of an imprinted domain from non-imprinted components. PLoS Genet. 2, e182 (2006). human disease research 101. Edwards, C. et al. The evolution of the imprinted Dlk1‑Dio3 domain in mammals. PLoS Biol. 6, e135 (2008). Timothy J. Aitman, Charles Boone, Gary A. Churchill, Michael O. Hengartner, 102. Renfree, M. B., Hore, T. A., Shaw, G., Graves, J. A. & Pask, A. J. Evolution of genomic imprinting: insights Trudy F. C. Mackay and Derek L. Stemple from marsupials and monotremes. Annu. Rev. Genomics Hum. Genet. 10, 241–262 (2009). 103. Moore, T. & Haig, D. Genomic imprinting in Abstract | Model organisms have played a huge part in the history of studies of mammalian development: a parental tug‑of‑war. human genetic disease, both in identifying disease genes and characterizing Trends Genet. 7, 45–49 (1991). 104. Keverne, E. B. & Curley, J. P. Epigenetics, brain their normal and abnormal functions. But is the importance of model organisms evolution and behavior. Frontiers in Neuroendocrinol. 29, 398–412 (2008). diminishing? The direct discovery of disease genes and variants in humans has 105. Rowe, H. M. et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, been revolutionized, first by genome-wide association studies and now by 237–240 (2010). whole-genome sequencing. Not only is it now much easier to directly identify 106. Martens, J. H. et al. The profile of repeat-associated histone lysine methylation states in the mouse potential disease genes in humans, but the genetic architecture that is being epigenome. EMBO J. 24, 800–812 (2005). 107. Watanabe, T. et al. Role for piRNAs and a novel RNA revealed in many cases is hard to replicate in model organisms. Furthermore, in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332, 848–852 (2011). disease modelling can be done with increasing effectiveness using human cells. 108. Glass, J. L., Fazzari, M. J., Ferguson-Smith, A. C. & Where does this leave non-human models of disease? Greally, J. M. CG dinucleotide periodicities recognized by the Dnmt3a‑Dnmt3L complex are distinctive at retroelements and imprinted domains. Mamm. Genome 20, 633–643 (2009). Why do we still need model organisms particular disease or trait. So establishing 109. Wallace, C. et al. The imprinted DLK1‑MEG3 gene region on chromosome 14q32.2 alters to understand human disease? the mechanism through which they act has susceptibility to type 1 diabetes. Nature Genet. 42, been elusive for all but a few GWAS hits. In 68–71 (2010). 110. Kong, A. et al. Parental origin of sequence variants Timothy J. Aitman. In May 2008, Nature addition, the environmental variation and that segregate with complex diseases. Nature 462, Genetics published a Focus issue on rat genet- outbred, heterogeneous genetic backgrounds 868–874 (2009). 1 111. Lubinsky, M., Herrmann J., Kosseff, A. L. & Opitz, J. M. ics. An article in that issue , co-authored of human studies reduce statistical power Autosomal-dominant sex-dependent transmission of and supported by over 250 rat geneticists, to detect gene effects, particularly trans- the Wiedemann-Beckwith syndrome. Lancet. 1, 932 (1974). outlined six principles that underline the case regulated effects (where sequence variation 112. Cattanach, B. M. & Beechey, C. V. in Chromosomes for continuing or even strengthening efforts at one locus acts by influencing gene expres- Today (eds Fredga, K., Kihlman, B. & Bennett, M.) 135–148 (Unwin Hyman, London, 1990). in animal genetics. These principles, which sion at a second locus that is remote from 113. Kanduri, C. et al. Functional association of CTCF with apply equally to other model organisms, the first), and gene–environment interac- the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive. Curr. Biol. include: the wealth of literature accumulated tions. Curiously, although gene loci identi- 10, 853–856 (2000). over 100 or more years for models such as the fied in human GWASs explain only a small Acknowledgements mouse and rat; the genome resources that, as proportion of the heritability of complex The author acknowledges the many scientists whose research for humans, have accelerated the pace of gene traits, such as body mass and blood pressure, has contributed to the genetic, embryological and epigenetic studies on parental-origin effects and genomic imprinting, identification for a wide range of phenotypes; studies in rodents explain several times the and apologizes to those whose work it has not been possible and the opportunities for in vivo phenotyp- total genetic variance for similar traits2,3. to mention or cite in the confines of this Timeline article. The author is grateful to previous and current members of the ing and gene targeting that have catalysed In the rat, integrating linkage analysis Ferguson-Smith team for their contributions to the ideas pre- our understanding of genetic mechanisms. with expression profiling has proved a par- sented here, and thanks the colleagues with whom she has spent many a long hour debating and discussing the past, However, genome-wide association ticularly powerful approach. The application present and future of genomic imprinting and epigenetic studies (GWASs) in humans have recently of this approach using adipose tissue led processes. identified hundreds of genes and gene loci to the identification of Cd36 as an insulin Competing interests statement for common human diseases. Furthermore, resistance gene in rats and humans4. This The author declares no competing financial interests. new sequencing technologies have acceler- was among the first complex trait genes

FURTHER INFORMATION ated the pace of discovery for Mendelian to be positionally cloned in any mammal. Anne C. Ferguson-Smith’s homepage: traits, providing insights into their molecular Building on this integrative strategy led to http://www.pdn.cam.ac.uk/staff/ferguson basis by direct study of the human diseases detection of thousands of rat expression Catalogue of Imprinting Effects: http://igc.otago.ac.nz/home.html rather than models. Surely it is not sufficient quantitative trait loci (eQTLs) in multiple Epigenesys (EU Network of Excellence): to continue studying animal models just tissues5,6 and identification of rat genes for http://www.epigenesys.eu International Human Epigenome Consortium: because it has always been this way? cardiac mass, cardiac failure, glomerulo- http://www.ihec-epigenomes.org One of the arguments in favour of con- nephritis and hypertension1,7, all of which MouseBook imprinting catalogue: http://www.mousebook. org/catalog.php?catalog=imprinting tinuing studies of model organisms is that show conserved function in humans. A high Medical Research Council Harwell Genomic Imprinting the genetic studies of common human dis- proportion of other complex trait genes homepage: http://www.har.mrc.ac.uk/research/ genomic_imprinting eases have significant limitations. The genes identified in the rat have conserved phe- University of Cambridge Centre for Trophoblast Research: and gene loci found by GWASs are mostly notypes in humans, in several cases more http://www.trophoblast.cam.ac.uk of small effect and explain a relatively low strongly so than corresponding mouse mod- ALL LINKS ARE ACTIVE IN THE ONLINE PDF proportion of the overall heritability for a els. Particular examples are: the polycystic

kidney disease gene Tmem67, as a cause of Although a comparable series of reagents is Of course, many fundamental insights one of the first defined ciliopathies; Fcgr3, not available for any other experimental sys- into basic aspects of cellular life — for exam- the first example of gene copy number vari- tem, yeast researchers have pioneered many ple, cell division, gene expression and signal ation as a cause of autoimmunity; Fbxo10 approaches that are generally applicable for transduction — have emerged from studies and Frmpd1, causes of oestrogen-responsive global surveys of gene function8,9. The yeast with yeast and have served as templates for breast cancer; and Apc, mutations in which community itself is in an arguably unique exploring these functions in human cells. cause colorectal cancer1. Examples of human position of having the potential to generate Indeed, there is tangible evidence that we biology revealed by rat genetics will continue a complete understanding of the biologi- are making major headway towards a gen- to increase in coming years, making a strong cal principles governing life at the level of a eral understanding of the individual roles case for continuing genetic studies in the rat. single cell. The relevance to human disease of each yeast gene, at least in a first-pass is self-evident: if we do not fully understand analysis. Most of the ~6,000 budding yeast Charles Boone. The budding yeast commu- the basic workings of a simple unicellular genes are now annotated with functional nity has spent the last decade or so producing eukaryote such as yeast, then how can we information in the Saccharomyces Genome tools and reagents that enable systematic anal- hope to decipher the more complex biology Database (SGD)10, as are many of the fis- ysis of gene function, resulting in an amazing of a normal human cell, let alone unravel the sion yeast genes in the Schizosaccharomyces catalogue of genome-wide strain collections. pathways that go awry in disease states? pombe GeneDB. So, do we know enough to

The contributors*

Timothy J. Aitman is Consultant Physician in the Imperial College apoptosis in this species. Michael has continued to work with C. elegans Healthcare National Health Service (NHS) Trust and Professor of Clinical ever since, first as a group leader at Cold Spring Harbor Laboratory, New and Molecular Genetics at Imperial College, both in London, UK, where York, USA, and for the last 10 years as a professor of molecular biology in he is Strategic Theme Leader for Genetics and Chair of the Imperial Zurich. Michael is a fellow of the American Association for the Molecular Pathology Group. He is a Fellow of the Royal College of Advancement of Science (AAAS), a member of the Deutsche Akademie Physicians and Academy of Medical Sciences and was the Specialist der Naturforscher Leopoldina and a recipient of the Swiss National Latsis Adviser for the House of Lords Science and Technology Committee’s Prize. Besides apoptosis, Michael is also interested in the evolution of 2009 Inquiry into Genomic Medicine. His research has combined the use proteomes and the regulation of mRNAs by microRNAs and RNA-binding of classical genetics with genome technologies to investigate the proteins. He is the founding chair of the Human Proteome Organisation genetics of common complex human disorders. These studies have led (HUPO) initiative on Model Organism Proteomes (iMOP) and co-initiator to the identification of Cd36 as an insulin resistance gene and Ogn as of the Basel Declaration. a cause of cardiac hypertrophy. They have also led to the finding that a new type of genomic sequence variation, gene copy number variation Trudy F. C. Mackay is a William Neal Reynolds Distinguished University at the Fcgr3 locus, is a cause of autoimmune glomerulonephritis and Professor of Genetics at North Carolina State University, Raleigh, USA. systemic lupus erythematosus. She received her Ph.D. in genetics from the University of Edinburgh, UK, in the laboratory of Alan Robertson and did a postdoctoral fellowship Charles Boone’s laboratory developed the synthetic genetic array (SGA) with Roger Doyle at Dalhousie University, Halifax, Canada. She has been method for automation of yeast genetic analysis. His group is applying at North Carolina State University since 1988. Her interests are the SGA on a large scale to map genetic-interaction networks as a global molecular genetic basis of variation for quantitative traits, and means of defining gene function and mapping a functional wiring mechanisms that maintain variation in complex traits within populations diagram of the cell. He holds a Canada Research Chair in Proteomics, and cause divergence of trait values between species. Bioinformatics and Functional Genomics and a Tanenbaum Chair at the University of Toronto, and he is a fellow in the Genetic Networks Program Derek L. Stemple is Acting Head of Mouse and Zebrafish Genetics at the of the Canadian Institute for Advanced Research. Wellcome Trust Sanger Institute, Cambridge, UK. His team works on a range of projects related to growth, development and human disease, Gary A. Churchill is a statistical geneticist at The Jackson Laboratory, using the model organisms Xenopus tropicalis and the zebrafish. Derek Bar Harbor, Maine, USA, where he has made major contributions to obtained his first degrees from the University of Colorado, Boulder, USA, understanding the genetics of health and disease using the mouse as a in applied mathematics (B.S.) and molecular, cellular and developmental model system. He contributed to the conception and implementation biology (B.A.). As a postgraduate, he worked for several years studying of the Collaborative Cross and the Diversity Outcross, which are new microtubule dynamics and mitosis with J. Richard McIntosh. He began his mouse populations for systems genetics. The Churchill laboratory has Ph.D. studies in neurobiology at the California Institute of Technology, generated a wealth of resources for mouse genetics, including: the first Pasadena, USA, under the supervision of David Anderson, which high-density genotyping platform for the mouse; the QTL Archive, a concluded with his discovery of the mammalian neural crest stem cell. As repository of genetic mapping data; the mouse SNP database; a Helen Hay Whitney postdoctoral fellow working with Wolfgang Driever R/maanova software for the analysis of gene expression microarray at the Massachusetts General Hospital, Boston, USA, Derek participated in data; and J/qtl software for QTL analysis. As director of the Center for a large-scale systematic screen for mutations affecting embryogenesis Genome Dynamics, a National Center for Excellence in Systems in zebrafish. As an independent investigator at the National Institute for Biology, he leads a consortium of investigators with a shared interest in Medical Research, London, UK, his group identified several genes systems genetics. important for development of the notochord. Currently, he is a senior group leader at the Wellcome Trust Sanger Institute, where his group Michael O. Hengartner is a professor at the Institute of Molecular Life studies the genetics of vertebrate early development, skeletal sarcomere Sciences and Dean of the Faculty of Science at the University of Zurich, formation and muscle integrity as well as zebrafish genomics. His group Switzerland. Michael was introduced to Caenorhabditis elegans as a is also involved with two major Sanger Institute projects: the Zebrafish graduate student in the laboratory of H. Robert Horvitz at the Mutation Project and the Zebrafish Sequencing Project. Massachusetts Institute of Technology (MIT), Cambridge, USA, where he contributed to the elucidation of the molecular machinery that controls *Listed in alphabetical order.

abandon yeasts, or do we still need them to Gary A. Churchill. The dramatic rate of This is the first reason why model organ- understand human disease? progress in human genetics has resulted isms are so useful. Even in years past, the I think all yeast researchers would agree from advances in genotyping technology, main benefit of model organisms was not to that we have much work to do before we can the mobilization of a research community facilitate the identification of human disease claim to fully understand even the best-char- committed to phenotyping large cohorts of genes or even to identify the specific func- acterized pathways, let alone how they are human subject and the development of new tion of disease genes but rather to put these wired together. Perhaps most importantly, statistical analysis methods13. New technol- disease genes into a biological context. We we almost completely lack a quantitative ogy — such as low-cost whole-genome study model organisms to understand life. understanding of most pathways and cannot sequencing — will continue to drive pro- A better understanding of a specific human even start to model most functions. Deep gress, but exclusive focus on human studies disease then simply comes as a consequence molecular insight is required to understand will ultimately limit the scope and power of of this better overall understanding of bio- disease gene biology, particularly with an eye biomedical research. The challenges posed logical processes. For example, the study of to developing targeted therapeutics. As such, by population stratification, rare alleles, Caenorhabditis elegans mutants that lack many ongoing studies of the precise roles genetic heterogeneity, uncontrolled environ- the ability to undergo developmental cell of yeast genes remain productive, especially mental variables and constraints on death led to the identification of caspases studies of those with homology to human experimental validation can be circumvented as apoptotic proteases16, and the analysis of disease genes. Below, I describe examples in model organisms. However, the full worms with small body size identified the that relate directly to human disease. potential of model systems, including the SMAD proteins as mediators of transform- Mutations in the highly conserved mouse, will only be realized if we re-evaluate ing growth factor-β (TGFβ) signalling17. Shwachman–Bodian–Diamond syndrome and modify our current strategies in light Perhaps the most unexpected discovery (SBDS) protein are associated with bone of the knowledge gained from the success of of all was that of microRNAs (miRNAs) marrow failure and leukaemia predisposi- human genetic studies. through the study of mutations that show tion in humans. However, only when Alan Disease alleles do not have to be discov- aberrant timing patterns during larval devel- Warren’s laboratory cracked its role in yeast11 ered in human populations in order to have opment in worms18,19 — hardly an obvious did we realize that SBDS is crucial for a relevance to human health. Model organisms place to search for what we now agree are key step in the translational activation of enable experimental interventions that can key disease modulators! The several Nobel ribosomes. One of the most amazing things establish causal mechanisms of gene action. Prizes in physiology and medicine that were about this discovery is that the human dis- They can provide unique genetic architec- conferred to model organism researchers ease phenotypes provided no obvious clues tures, such as inbred strains and isogenic over the last two decades nicely underscore about the underlying molecular defect. lines that are ideal for investigating genetic the success of this approach. Studies in an experimentally tractable sys- interactions with the environment. Studies The second major advantage of model tem that enables unbiased genetic experi- of potentially harmful interventions require organisms is that they can be manipulated ments were required to solve the problem. model systems. The similarity of genetic experimentally much more readily than This will continue indisputably to be the and physiological mechanisms shared by humans because of both ethical and tech- case as the genes underlying more human all mammals has empowered the use of the nical issues. Many important questions diseases are discovered, particularly when laboratory mouse to identify genes and to simply cannot be addressed adequately if complex genetic interactions are involved in investigate genetic mechanisms that are you are restricted to using patients as your the disease phenotype. relevant to the prevention and treatment of ‘experimental system’. Particularly compel- In April 2010, the New York Times human disease for more than a century14,15. ling methodological contributions from the published an article entitled ‘The Search The full potential of the mouse as a model worm field include the development of gene for Genes Leads to Unexpected Places’. system is still to be realized. knockdown via RNAi20 and the development The opening paragraph describes how Ed of green fluorescent protein (GFP) as an Marcotte’s laboratory developed a compu- Michael O. Hengartner. New sequencing in vivo reporter21 (resulting in Nobel Prizes in tational approach to define precise gene technologies indeed promise a new golden physiology and medicine in 2006 and models for human diseases12. They estab- age for human genetics. Will this golden age in chemistry in 2008). lished a database of hundreds of associa- spell doom for model organisms? Most Of course, one can also work with human tions between genes and human diseases, certainly not. Quite the contrary: I am cell lines. These can be manipulated easily, and then they integrated thousands of other convinced that it will, in fact, lead to an and work on cell lines has contributed associations between these genes and mutant increased need for model organism research. greatly to our understanding of human phenotypes in several model organisms. Many new human disease genes and biology. But work with cell lines has its own The resultant network revealed that mouse candidate disease genes will be identified in limitations, particularly when studying genes that are known to help build blood coming years. But why do mutations in these processes at the tissue, organ or whole-body vessels were closely related to yeast genes genes cause disease and what biological pro- level. Thus, human cell lines can certainly involved in stress response signalling and cesses are affected? Sequence variations will complement model organisms but cannot cell wall biogenesis. Thus, some creative not give us answers to these questions; only fully replace them. network analysis identified an unintuitive biological investigations will. Even today, we Let me end by noting that this debate has, yeast model for angiogenesis, illustrating the only understand the biological function of in fact, been going on for a while already, potential for an untapped wealth of molecu- a fraction of human genes. Linking a poorly albeit in another forum. The question of lar insights about human diseases that characterized gene to a human disease does whether model organisms are still needed is remain to be discovered in yeast and other not per se make this gene better understood raised regularly by members of the animal model systems. — it just makes it ‘more interesting’. rights movement, who suggest that, in the

context of biomedical research, experiments affecting quantitative traits can be derived. In experimental terms, the genetic com- on animals (in this case, mainly mammals) QTLs can be mapped with high resolution plexity underpinning quantitative traits are no longer useful and can today be using complementation tests to deficiencies in flies and humans necessitates a shift in replaced by in vitro experiments, cell lines and mutations. Most recently, GWASs have focus from the individual locus to genetic and computer simulations. While efforts been performed for organismal and gene- networks. This is readily accomplished by in this direction are certainly not only pos- expression traits using a reference popula- using enhancer and suppressor screens sible but also desirable (see, for example, tion of fully sequenced inbred fly lines in in flies that ectopically express human the ‘3R principle’ of replacement, refine- which local LD decays in less than 200 bp23,24. transgenes to identify interacting loci31,32. ment and reduction and the recent Basel These studies have revealed that hundreds It can also be achieved by using systems- Declaration22), life scientists and regulatory of loci typically affect each trait and that genetics24,33,34 approaches to construct causal agencies agree that animal experiments are synonymous polymorphisms and poly- transcriptional networks associated with absolutely essential. morphisms in putative regulatory regions natural variation in complex traits. While From both points of views, model organ- more commonly affect complex traits than the systems-genetics approach is possible isms are thus — at least for the foreseeable non-synonymous polymorphisms. Thus, the in humans, it is limited by the difficulty in future — truly ‘irreplaceable’. genetic architecture of human complex traits obtaining samples of appropriate tissues that and diseases was not only replicated but was are relevant to disease35,36 and the challenges Trudy F. C. Mackay. Despite the advances also anticipated from studies in a model of testing model predictions. in identifying genetic variants associated organism. with human disease, we must remain cog- There is also more to be learned from Derek L. Stemple. The basic biological nizant that genetic research in humans is a studies of genetic architecture in the fruitfly. processes that underlie many pathologies descriptive, hypothesis-generating endeav- The loci implicated in D. melanogaster are very difficult to determine when only our. The resolution of human GWASs is quantitative traits are typically novel, com- human data are considered. limited by the scale of linkage disequilibrium putationally predicted genes and genes with First, the data collected for human (LD) — any locus within a large LD block unexpected effects on the trait of interest, disease studies are inherently limited to pro- associated with a trait could potentially highlighting our poor understanding of the cedures that are appropriate to carry out in be causal, and identifying causal variants biology that underlies complex organismal humans. The kind of detailed studies needed within a gene cannot be accomplished using phenotypes. Our recent fly GWASs indicate to understand the basic biology of disease, population data. Determining causality can a ‘J-shaped’ distribution of allelic effects for however, often involve the intentional gen- sometimes be inferred from functional stud- all traits, whereby the alleles with the larg- eration of pathological states to allow the ies in cell lines, but more often it requires est effects are at low frequency, and those controlled measurement of the direct out- studies of the effects of mutations or human with the smallest effects are at intermediate come of specific lesions. For example, studies transgenes in model organisms. Model frequency. Finally, sex- and environment- of the functional effects of genetic variants organisms also play a central role in charac- specific effects and epistasis figure promi- carried out directly in humans may provide terizing the general features of the genetic nently in the genetic architecture of most some information. However, such studies are architecture of quantitative traits, which D. melanogaster quantitative traits23,24. generally limited to traits that can be meas- also apply to human quantitative traits and Such observations are relevant in explain- ured in a relatively non-interventional fash- diseases. ing the ‘missing’ heritability in human ion, which tend to be those such as blood In terms of functional annotation, many GWASs25 and have implications for the pressure that involve highly integrated pro- canonical signalling pathways affecting future of personalized medicine. If the distri- cesses. More direct measurements or experi- human disease (such as Notch, Wingless, bution of allelic effects for human complex mental perturbations are usually not possible Hedgehog and epidermal growth factor) traits is similar to that observed in flies, then in humans. So while the proximate cause of were originally described in Drosophila GWASs in humans have only identified the high blood pressure may be an increase in melanogaster. Thus, using the power of intermediate frequency alleles with small circulating levels of a hormone or cytokine, fruitfly genetics to systematically charac- effects — low-frequency causal variants the mechanism behind these increased levels terize pleiotropic effects of mutations and with larger effects are poorly tagged by the may be a small group of cells, such as specific natural variants on organismal phenotypes intermediate-frequency SNP markers that hypothalamic neurons, which either secrete relevant to human biology will provide novel are used in human GWASs. Furthermore, the hormone or cytokine or indirectly regu- annotations to inform interpretation of sex-specific effects26 and genotype–environ- late its secretion. Animal studies combined human GWASs. ment interactions27–29 affecting human dis- with human studies allow us to tease apart Studies of the genetic architecture of eases and quantitative traits have been found complex mechanisms, as the relevant tissue complex traits in D. melanogaster benefit in the rare attempts that have been made to from the animal can be interrogated at the from several features. The genetic back- look for them. When these effects exist but appropriate time and context to understand ground and environment can be precisely are not accounted for, this contributes to the disease. controlled and replicated, and quantitative missing heritability. Finally, epistasis is gen- Second, although there has been huge phenotypes can be precisely defined and erally ignored in human association studies progress in medical imaging, it is often not measured at high-throughput. The effects of for several reasons30, but could be crucial possible in humans to achieve cellular and de novo mutations on multiple quantitative to personalized medicine — averaging the subcellular resolution of important events traits, including whole-genome transcript effects of individual SNPs across extensive that are relevant to disease. With model abundance, can be assessed. Epistatic inter- pairwise and higher-order epistatic interac- organisms, it is possible to tag normal actions among these mutations can be pre- tions in human GWASs could also contribute cellular proteins by fusion with fluorescent cisely quantified and hence genetic networks to the observed small effect sizes. proteins. Using such techniques, researchers

have made great strides in understanding the multiple omics data sets being generated by transformative toolkit for disease gene dynamics of gene expression, protein activi- EURATRANS, these resources are antici- identification and deep understanding of ties and cell fate using transgenic animals. pated to catalyse a systematic elucidation mechanisms underlying pathobiology. This is nicely exemplified by the multiplex of the molecular basis of scores of disease expression of different fluorescent proteins phenotypes that have been mapped to the rat C.B. The role that yeast plays in our under- in transgenic mice to map connectivity in genome as QTLs over the last two decades. standing of human disease goes far beyond the developing brain37. Increasingly, such Following more than 20 years in which that of a functional catalogue of its parts49. studies involve the real-time live imaging of targeted knockouts have been possible in When our understanding of human disease biological processes that would be essentially the mouse and not the rat, two gene-targeting genetics becomes more sophisticated, so impossible in humans. Moreover, some techniques have recently been pioneered does our potential to use the yeast model model organisms (such as zebrafish) enable for the rat. First, using new protocols for system for studying the underlying molecu- fluorescent probes to be combined with generating rat embryonic stem cells, p53 lar mechanisms. In addition, yeast can be the real-time imaging of important events. knockout rats have been created by homolo- used as a surrogate cell to model disease For example, real-time live-imaging studies gous recombination45, paving the way for states that often appear to be decidedly in zebrafish have provided important new conditional and tissue-specific knockout ‘non-yeast’ at first glance. insights into blood vessel development38. rats. Second, zinc finger nucleases have Budding yeast can grow without mito- A third key contribution of model organ- been shown to be highly efficient at creating chondrial respiration, providing a unique isms comes from a genetic point of view. In gene-targeted rat knockouts46. With funding model for revealing disease genes associ- the human population as a whole there is from the US National Institutes of Health ated with this important organelle50. Jared likely to be an exhaustive coverage of both to create new gene knockout models for rat Rutter’s laboratory discovered that the yeast single-nucleotide and structural genetic vari- genes orthologous to human GWAS hits, SDH5 gene (also known as EMI5), which is ation in the coming years. However, making 54 rat gene knockouts have been created in the homologue of the PGL2 gene mutated use of this information to understand the past year47. Some of the rat knockouts in the hereditary cancer paraganglioma, disease would require complete or near- that have already been phenotyped show is required to modify key respiratory complete sequencing of the DNA of every greater similarity to human disease traits proteins51. Susan Lindquist’s laboratory human, combined with a detailed personal than corresponding mouse knockouts. is creatively studying Alzheimer’s and health and trait history. With model organ- These rat models are anticipated to give Parkinson’s diseases through heterologous isms, it is possible to vastly increase the rate significant new insights into the biological expression of the human disease proteins of mutation discovery and phenotype analy- mechanisms underlying genes identified in in yeast cells52. These neurodegenerative sis. The use of efficient chemical mutagens, human GWASs. diseases are associated with protein mis- insertional mutagens and mutagens that lead The rat genetics community has placed folding, oligomerization and aggregation, to structural rearrangements are standard a high priority on translating biological processes that are investigated readily by practice in model organism research. Our insights gained in the rat to advance under- hijacking the highly conserved protein current understanding of the genetics con- standing of human diseases, and many quality-control system in yeast. In another trolling metazoan development is derived rat QTL genes identified to date have example, Jasper Rine’s group is investigating largely from such random mutagenesis been shown to have conserved functions cofactor-dependent enzymes, such as the studies in worms, flies and zebrafish39–42. in humans1. Recently, a systems-genetics vitamin-dependent enzyme methylene- approach carried out primarily in the rat tetrahydrofolate reductase (MTHFR), by How is model organism research revealed one of the first trans-regulated complementation analysis of the relevant adapting to the changing landscape mammalian expression networks, a goal yeast mutant with a variety of human vari- of disease genetics? that has proved difficult in humans. The ants53. In doing so, they ask yeast to tell us network is driven by a common regulator, which human variants are compromised for T.J.A. Genome technologies have driven Ebi2 (also known as Gpr183), which is con- function and which of these are subject to major progress in human genetics in the served in rats and humans, is expressed in vitamin remediation. mid to late 2000s. Similar opportunities have macrophages and is associated in GWASs In my opinion, one of the most promising not passed the rat genetics community by with human type 1 diabetes48. Such systems- avenues of future research that is relevant to unnoticed. genetics studies are possible in rats because the changing landscape of disease genetics is With funding from two European Union of the ready availability of ex vivo tissues and investigating how an individual’s phenotype consortium awards, EURATools and the statistical power gained from studies of is dictated by their genotype. The awesome EURATRANS, more than 15 rat genomes inbred strains in controlled environments. power of yeast genetics and its relatively have been sequenced using next-generation Overall, these vignettes provide clear small haploid genome makes it one of the platforms, including upgrades to the brown examples of the translational focus of the simplest and most effective model systems Norway reference genome (E. Cuppen, rat genetics community in an era of unprec- for studying the genetic diversity that exists Utrecht, personal communication; edented scientific opportunity enabled within a species. The challenge becomes one N. Hubner, Berlin, personal communication; by ultra-high-throughput genomics and of developing efficient methods for analysing T.J.A. unpublished observations) and mathematical biology. While the mouse complex QTLs on a large-scale, such as the sequencing and characterization of the will remain an important model system for extreme QTL (X-QTL) mapping approach genome of the spontaneously hypertensive functional studies, the genetic, genomic and invented by the Kruglyak laboratory54. Our rat43. Along with extensive eQTL and phe- gene-targeting techniques now available in global mapping of synthetic lethal genetic notype resources in recombinant inbred and the rat, when integrated with human GWASs networks55 highlights the importance of heterogeneous stock panels5,6,44, as well as and deep-sequencing approaches, provide a genetic interactions in determining the

phenotype for a given individual. As articu- Absence of population structure limits knowledge gained from the model system lated by Lee Hartwell and colleagues56, one the risk of finding spurious associations. should be used to inform further studies hypothesis is that genetic interactions among Balanced allele frequencies — in particu- that can be carried out directly on human personal allelic variants may underlie much lar, the absence of rare alleles — maximize subjects. I would assert that studies using of the genetic architecture that dictates a statistical power. Availability of complete mouse models will most often lead to valid given phenotype57, including genetic dis- genome sequences enables unambiguous and useful insights that will advance bio- eases. Over the next decade or so, the analy- haplotype reconstruction, which allows for medical practice at a faster pace and at a sis of the phenotypes associated with natural the application of powerful linkage-analysis considerably lower cost than can be achieved variation in yeast may very well provide methods. A model organism population by other approaches. We need both human important insights into the ‘missing herit- with all of these properties could achieve and model systems to make progress. Each ability’ problem that has been implicated by greater statistical power and higher map- approach complements the other and human GWASs25. ping resolution compared to a human neither alone will suffice. genetic study, with substantially lower cost G.A.C. The same technologies that have and smaller sample size. With human study M.O.H. Model organism research is indeed enabled advances in human genetics can be sizes already soaring into tens of thousands evolving — not necessarily owing to the applied in model organisms58. New technol- and beyond, at some point we will reach a changing landscape of human disease ogy can also provide approaches that reach practical limit. genetics but rather in response to the same beyond the potential of human genetics. The Recognition of the shortcomings of drivers. The new sequencing technologies predominance of the mouse in biomedi- existing mouse genetics resources preceded that are revolutionizing human genetics cal research can be attributed in part to the the GWAS era64 and led to the development are having a similar effect in the model development of targeted gene replacement of the Collaborative Cross, a new collection of organism communities with ‘thousand in embryonic stem cells15. This technology inbred strains with unprecedented levels genome projects’ for model organisms and has played a central role in forward genetic of genetic diversity65,66. The first fully inbred whole-genome sequencing of mutant col- approaches that aim to establish gene func- Collaborative Cross strains are completed lections, large-scale projects to collect and tion, and there is an ongoing effort to create and the panel is expected to grow to more study sequence variation — be it natural or a comprehensive panel of single-gene con- than 300 unique inbred strains within a few induced — abound. As has been the case ditional knockout alleles59. Although fixed years. The Diversity Outcross is an outbred with single-gene analyses, the goal of these genetic background and the extreme nature population derived from the same founders studies is not necessarily to model human of the genetic perturbation limits their value as the Collaborative Cross. The breeding disease per se but to better understand as models of complex disease, knockout designs of these populations maintain bal- the relationship between genotype and mice could be employed to map genetic anced allele frequencies and minimize pop- phenotype; for example, how robustness interactions60 and to identify modifier loci61, ulation structure. The same set of 45 million is generated in signalling pathways70,71 or providing new insights into mechanisms and SNPs and an estimated 4 million structural how regulatory processes can evolve over potential modes of intervention. variants segregate in the Collaborative Cross micro-evolutionary times72. The natural history of Mus musculus and Diversity Outcross strains, which have The second general driver is the desire is distinct from that of humans and, as distinct and complementary genetic archi- and need to integrate multiple data types. such, mice have little to offer as a model of tectures. The Collaborative Cross provides Model organisms can also help on this human population structure62,63. However, fixed, reproducible genomes, whereas the front by serving as smaller ‘pilot projects’, there is no reason to replicate human-like Diversity Outcross provides an unlimited for example. Only by integrating genomics demographics in a model system. To illus- number of unique allelic combinations and with other omics data types can we hope to trate this point, consider rare alleles. From a mapping resolution that will soon exceed ultimately truly understand and predict how the perspective of an individual who carries that which is attainable in human popula- changes in the genome and epigenome affect a disease-causing allele, its frequency tions. By design, these populations eliminate the phenome at the molecular, cellular and is irrelevant. What really matters for the many of the pitfalls that hamper progress organismal levels. The recent publications prevention or treatment of disease is in human genetic studies. The Diversity from the C. elegans and D. melanogaster the nature of the allelic effect, its mode of Outcross and Collaborative Cross provide modENCODE consortia73,74 give a feeling action and its potential interactions with a complementary system for discovery and of what is possible in this respect and nicely environmental factors. Rare alleles, even validation that will help to usher in a new demonstrate that model organisms still have those of large effect, can be exceedingly era of experimental genetics67. much to offer. difficult to identify in natural populations. Experimental intervention is the funda- It is clear that big collaborative science It would not make sense to intentionally mental scientific method used to establish has also come to the small model organisms, create a model system with rare alleles. causality. Despite the high precision of map- and it is highly unlikely that this trend will Artificial populations can be designed to ping in GWASs, the identification of causal end any time soon. Databases will continue provide a more effective basis for discovery variants presents significant challenges68,69 to increase in number, size and complexity. of disease-causing alleles and elucidation of and subsequent experimental validation Computer and bioinformatic literacy, still the mechanisms of disease causation. demands the use of model organisms. much too often delegated to an expert So what population characteristics are Whether it is the discovery or validation of collaborator, will need to become part of desirable in a genetic model system? High a causal allele that is achieved in a mouse everybody’s toolkit. In the future, scientists genetic diversity maximizes the opportunity model, insights into the human condition working on model organisms will need not for discovery. High mapping resolution leads will, of course, require extrapolation beyond only a ‘feeling for the organism’ (REF. 75) but to efficient identification of causal genes. the inferential scope of the experiment. The also a ‘feeling for the algorithm’.

T.F.C.M. There have been limitations in can rapidly provide valuable information on a wide variety of backgrounds is now using the fruitfly to study the involvement of about the suspected disease gene78. Indeed, possible using zinc finger nuclease technol- naturally occurring genetic variation in com- the combination of morpholino oligonu- ogy, which may be applied, for example, in plex genetic traits. Genotyping and sequenc- cleotide knockdown with overexpression of congenic rat strains with susceptibility to ing costs are the same in D. melanogaster as wild-type and mutant forms of the suspected common diseases, such as hypertension or in humans. Furthermore, the high level of disease gene can provide both the necessary diabetes47. polymorphism and rapid decline in local LD control for the morpholino oligonucleotide Timothy J. Aitman is at the Physiological Genomics in D. melanogaster preclude the development and subtler information concerning the phe- and Medicine Group, Medical Research Council of a common genotyping platform with the notypes that are produced by hypomorphic Clinical Sciences Centre, Imperial College, cost amortized across the community, as has variants of the gene79. Ducane Road, London W12 0NN, UK. been done for human genotyping platforms. For common diseases, a wealth of genetic e-mail: [email protected] As such, the extensive natural genetic varia- loci have been identified that explain Charles Boone is at the Banting and Best Department tion in fruitfly complex trait phenotypes has variable proportions of the heritability of a of Medical Research and the Department of not been efficiently explored, since the cost number of diseases80. The loci that underlie Molecular Genetics, Donnelly Centre, University of Toronto, 160 College Street, Toronto, greatly exceeds the budget of any individual common diseases can be within annotated Ontario M5S 3E1, Canada. research grant. However, the rapid advances genes but often lie in putative regulatory e-mail: [email protected] in next-generation sequencing technology regions. Again, loss-of-function model Gary A. Churchill is at The Jackson Laboratory, and equally rapid decline in costs have been organism data can provide good information 600 Main Street, Bar Harbor, game changers in model organism complex about whether a candidate variant is likely to Maine 04609, USA. trait genetics. These advances have stimu- contribute to a particular disease. But there e-mail: [email protected] lated the development of the Drosophila are several other important applications Michael O. Hengartner is at the Institute of Molecular Genetic Reference Panel24 and the mouse of model organisms in this respect. Using Life Sciences, University of Zurich, Winterthurerstrasse Collaborative Cross76 — mapping popula- comparative genomics, the analogous regu- 190, CH‑8057 Zurich, Switzerland. e-mail: [email protected] tions with high genetic diversity in which all latory regions between humans and mice molecular variants are known and that are can be identified, and appropriate deletions Trudy F. C. Mackay is at the Department of Genetics, North Carolina State University, Raleigh, available to the entire fly and mouse com- or sequence variants can be made in a mouse North Carolina 27695, USA. munities. With these resources in place, all model. It is also possible in model organ- e‑mail: [email protected] individual researchers need to do is use their isms to generate and combine specific alleles Derek L. Stemple is at the Wellcome Trust Sanger ingenuity to develop assays for phenotypes in multi-homozygous, heterozygous and Institute, Wellcome Trust Genome Campus, relevant to human diseases and quantitative mixed states to emulate the combinations Hinxton, Cambridge CB10 1SA, UK. traits, screen the mapping population and of variation that are causative for disease in e‑mail: [email protected] identify associated molecular variants. The humans. The effects of combining alleles for doi:10.1038/nrg3047 power of model organism genetics can a particular pathogenic trait are not easily 1. Aitman, T. J. et al. Progress and prospects in rat then be used to quickly map causal variants predicted and are not likely to reflect linear genetics: a community view. Nature Genet. 40, and transcriptional genetic networks. increases in severity; such nonlinearity has 516–522 (2008). 81 2. Shao, H. et al. 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