Research Collection Doctoral Thesis A systems genetics approach to understanding intra-species variation in Drosophila wing and body size Author(s): Vonesch, Sybille Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010419387 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 22296 A systems genetics approach to understanding intra-species variation in Drosophila wing and body size A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by Sibylle Chantal Vonesch MSc Universität Zürich born on 11.10.1984 citizen of Dübendorf ZH accepted on the recommendation of Prof. Dr. Ernst Hafen Prof. Dr. Konrad Basler Prof. Dr. Sven Bergmann Prof. Dr. Trudy F. C. Mackay 2014 1 1. SUMMARY 5 2. ZUSAMMENFASSUNG 7 3. INTRODUCTION 9 3.1. Animal Growth is a regulated process that is sensitive to environmental fluctuations 9 3.2. Molecular mechanisms governing growth control 10 3.2.1. The insulin/IGF pathway controls cell, organ and organismal growth and metabolism in response to nutrient availability 12 3.2.2. Insulin is the main regulator of metabolic homeostasis in vertebrates 13 3.2.3. Insulin-like growth factors (IGFs) control pre- and postnatal growth in vertebrates 13 3.2.4. Insulin and IGF binding to their corresponding receptors initiates an intracellular signaling cascade 14 3.3. Molecular mechanisms of growth control in Drosophila 16 3.3.1. Systemic control of growth and metabolic homeostasis by IIS/TOR signaling in Drosophila in response to nutrients 16 3.3.2. The transcription factor FOXO regulates many downstream targets of IIS and is essential for survival under stress conditions 20 3.3.3. The Target of Rapamycin (TOR) pathway 21 3.3.3.1. The TOR pathway controls cellular and organismal growth in response to local abundance of growth factors, amino acids, cellular energy levels and stress 21 3.3.3.2. TORC1 responds to growth factors via IIS, extracellular amino acid levels, intracellular energy status and stress 22 3.3.3.3. TORC1 promotes translation, ribosome biogenesis and the expression of metabolic genes while inhibiting autophagy 24 3.3.4. Myc controls cell growth and cell numbers downstream of TOR and other pathways 26 3.3.5. The Hippo tumor suppressor pathway controls cell number by regulating cell cycle progression and apoptosis in an organ autonomous manner 28 3.3.6. The Ras/MAPkinase pathway controls cell growth and proliferation 30 3.4. Steroid hormones affect body size by controlling developmental timing in Drosophila 32 3.5. Various environmental factors can influence body and organ size in Drosophila 34 3.6. The Drosophila wing as a model system to study growth control 36 3.7. Genome-wide association studies 40 3.7.1. Genome-wide association studies provide a first step towards a systems 2 level understanding of multigenic traits by linking variation in genotype to natural phenotypic variation 40 3.7.2. Finding the missing heritability of complex traits 45 3.7.3. GWAS studies of human height imply common variants with small effects account for half of the total heritability and shed light on the sources of missing heritability 46 3.7.4. Limitations and problems of human GWAS that can be overcome in model organism GWAS 48 3.7.5. Drosophila as a model system for GWAS of size 50 3.8. Experimental evolution 50 3.8.1. Experimental evolution experiments can be used to generate extreme phenotypes and to identify combinations of loci that are causally linked to these phenotypes 50 3.8.2. Results from selection studies of size in Drosophila 53 3.9. Summary of the introduction and project motivation 54 3.10. Aims of this thesis 55 4. RESULTS 57 4.1. Novel loci rather than variants in canonical growth pathway genes are associated with wing and body size variation in Drosophila melanogaster (Manuscript in preparation) 59 4.2. The FlyCatwalk: A high throughput feature-based sorting system for artificial selection (Submitted manuscript) 109 4.3. Further results 130 4.3.1. Foodbatch variability is a strong and specific confounder for wing size in Drosophila melanogaster 130 4.3.2. Experimental evolution of Drosophila wing size 133 5. DISCUSSION AND OUTLOOK 136 5.1. Small fluctuations in foodbatch quality cause substantial population-level variation in wing size 136 5.2. Many loci with small effects and predominantly regulatory variants underlie wing size variation in Drosophila 141 5.3. Novel candidates fall into diverse functional classes, overlap candidates from other studies and are associated with height or obesity related traits in humans 146 5.4. Planar cell polarity (PCP) genes and growth control 148 5.5. Metabolism and growth control 151 5.6. Conclusions and Outlook 154 3 6. MATERIALS AND METHODS 161 7. REFERENCES 162 8. ACKNOWLEDGEMENTS 200 9. CURRICULUM VITAE 201 4 1. SUMMARY All animals must regulate growth in a coordinated manner. A deregulation of this process impacts on survival, fitness and fertility of the organism and may lead to one of many malignancies subsumed as cancer. For understanding both physiological development and malignant growth it is thus paramount to gain a complete understanding of the mechanisms and genetic networks underlying growth control. The final size of an organism is determined by the interplay between intrinsic and extrinsic factors. Intrinsic factors are the genomic loci that exert their effect on growth via a highly interconnected network of cellular signaling molecules and systemic growth factors and hormones. The best-studied extrinsic factor is nutrient availability, but other factors such as temperature, population density and parasitic infection can also influence growth. The actions of intrinsic and extrinsic factors must be coordinated during development to ensure that growth can be adjusted to changing environments, for example in response to nutrient shortage. Much has been learned about the control of organismal and tissue growth from single gene studies in model organisms, especially Drosophila. On the systems level there are growth factors, which enable integration of growth with nutritional cues. The evolutionarily conserved insulin/insulin-like growth factor (IIS) and target of rapamycin (TOR) pathways are the main mediators of growth and proliferation in response to nutrient availability. On the level of the individual organs, morphogens and physical forces act to control size. Organs also have differential sensitivity to environmental cues, indicating that integration with the external environment not only occurs at the systemic but also at the organ level. On the cellular level, multiple pathways affect cell growth, proliferation and apoptosis in response to systemic and organ intrinsic stimuli and need to be integrated with each other. Besides growing, tissues also have to become patterned to form a normally developed organism, which involves dedicated pathways that may also impact on growth. On top of the molecular machinery is a layer of hormonal control that delimits the duration of different developmental stages. In light of the multifactorial nature of animal growth it is clear that regulatory networks rather than single genes or pathways govern the control of growth and proper organismal size. To gain a complete understanding of growth control we thus have to apply methods that probe the influence of whole gene networks and eventually the whole genome, rather than single genes, on size. It will ultimately be important to understand how all these genes interact under natural circumstances to create a properly sized organism, and how genetic variation creates phenotypic variability while preserving function. Exploiting natural phenotypic variation in size and studying which genetic loci cause this variation is a promising new approach for shedding more light on growth control at the systems level. Genome-wide 5 association studies (GWAS) are a routinely used tool for studying genotype phenotype associations in a variety of organisms and have substantially broadened our understanding of the underlying genetic networks of multifactorial traits. This work describes the application of genome wide association methods towards identifying the loci underlying natural wing and body size variation in Drosophila. We successfully reduced the influence of environmental factors during growth by raising flies under a strictly controlled regime, which is a necessity to avoid false associations between genotype and phenotype. We show that only very few of the previously known growth pathway genes are associated to variability in size, instead our study identifies novel regulators of size. This finding illustrates the complementarity of the GWAS approach to classical genetics and highlights the importance of probing natural variants. Our findings suggest processes like planar cell polarity and metabolism may have a larger role in controlling growth than previously thought. Furthermore, our results highlight the importance of intergenic noncoding and regulatory elements in creating size variability in a population and encourage more efforts towards the investigation of regulatory rather than functional mutations for understanding how phenotypic variability is achieved. Taken together our findings identify loci relevant for creating variability in size between organisms and add to the expanding knowledge of the processes governing