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  1. 1. Model Organisms Jennifer Slade B.Sc (Hon), M.Sc Candidate
  2. 2. Outline <ul><li>Model organism </li></ul><ul><ul><li>Definition </li></ul></ul><ul><ul><li>Current models </li></ul></ul><ul><ul><li>Characterisitics of a “good” model organism? </li></ul></ul><ul><li>Drosophila as a model organism </li></ul><ul><ul><li>Characteristics </li></ul></ul><ul><ul><li>Uses in Research </li></ul></ul><ul><li>Developmental disorders </li></ul><ul><ul><li>Conserved genes, similar functions </li></ul></ul><ul><ul><li>Conserved genes, different functions </li></ul></ul><ul><li>Neurological disorders </li></ul><ul><ul><li>Triple-repeat diseases </li></ul></ul><ul><ul><li>Parkinson disease </li></ul></ul><ul><ul><li>Familial Alzheimer disease </li></ul></ul><ul><ul><li>Fragile X </li></ul></ul><ul><li>Cancer </li></ul><ul><ul><li>RTK-RAS-MAPK signaling </li></ul></ul><ul><ul><li>Targets of Rapamycin pathway </li></ul></ul><ul><ul><li>Cell cycle control </li></ul></ul><ul><ul><li>Tumour metastasis </li></ul></ul><ul><li>Limitations of fly models </li></ul><ul><li>Summary </li></ul>
  3. 3. Definition of Model Organism <ul><li>Specific species or organism </li></ul><ul><li>Extensively studied in research laboratories </li></ul><ul><li>Advance our understanding of </li></ul><ul><ul><li>Cellular function </li></ul></ul><ul><ul><li>Development </li></ul></ul><ul><ul><li>Disease </li></ul></ul><ul><li>Ability to apply new knowledge to other organisms </li></ul>
  4. 4. Current Models <ul><li>Drosophila </li></ul><ul><li>Xenopus </li></ul><ul><li>Zebrafish </li></ul><ul><li>Mouse </li></ul><ul><li>C. elegans </li></ul><ul><li>Yeast </li></ul><ul><li>E. coli </li></ul><ul><li>Arabidopsis </li></ul>
  5. 5. Characteristics of a “Good” Model Organism <ul><li>Think individually </li></ul><ul><ul><li>Make jot notes </li></ul></ul><ul><ul><li>5 to 10 minutes </li></ul></ul><ul><li>Share in groups </li></ul><ul><ul><li>Get in groups of 4 </li></ul></ul><ul><ul><li>Discuss various characteristics </li></ul></ul><ul><li>Share with the class </li></ul><ul><ul><li>One person from each group write one characteristic discussed on board </li></ul></ul><ul><ul><li>Explain why characteristic is beneficial </li></ul></ul>
  6. 6. Drosophila melanogaster as a model organism
  7. 7. Characteristics of Drosophila that make it a good model organism <ul><li>Small, easy and cheap to maintain and manipulate </li></ul><ul><li>Short lifespan </li></ul><ul><li>Produce large numbers of offspring </li></ul><ul><li>Development is external </li></ul><ul><li>Availability of mutants </li></ul><ul><li>Lots of history/previous experiments and discoveries </li></ul><ul><li>Genome is sequenced </li></ul><ul><li>Homologues for at least 75 % of human disease genes </li></ul><ul><li>Exhibit complex behaviours </li></ul><ul><li>Fewer ethical concerns </li></ul>
  8. 8. Drosophila in Research <ul><li>Early research aided in the understanding of development </li></ul><ul><ul><li>Made first link between chromosome and phenotype </li></ul></ul><ul><ul><li>Identified various genes and mechanisms of development </li></ul></ul><ul><li>Current research focuses on the study of human disease </li></ul><ul><ul><li>Developmental disorders </li></ul></ul><ul><ul><li>Neurological disorders </li></ul></ul><ul><ul><li>Cancer </li></ul></ul>
  9. 9. Technique: Second site modifier screen <ul><li>Begin with a fly posessing a mutant phenotype </li></ul><ul><li>Create random mutations that might effect this phenotype in this genetic background </li></ul><ul><ul><li>Via radiation or feeding of a mutagen </li></ul></ul><ul><ul><li>Observe offspring or “grandoffspring” for either less or much more severe phenotype </li></ul></ul><ul><li>Some might be revertants of the original gene </li></ul><ul><li>Others might be mutants for upstream or downstream components of the pathway(s) </li></ul><ul><li>that lead to the original phenotype </li></ul><ul><li>Rarely, there might be mutants of a gene </li></ul><ul><li>with a compensational function. </li></ul><ul><li>These are second site mutants. </li></ul>
  10. 10. Human Disease : Developmental Disorders <ul><li>Dysmorphologies </li></ul><ul><ul><li>Diseases resulting in morphological defects </li></ul></ul><ul><ul><li>Largest, most prevalent human genetic disorders </li></ul></ul><ul><li>Result from mutations in genes that control important steps in development, such as: </li></ul><ul><ul><li>Transcription factors </li></ul></ul><ul><ul><li>Proteins involved in signal transduction </li></ul></ul><ul><li>Two broad categories: </li></ul><ul><ul><li>Conserved genes with orthologous function </li></ul></ul><ul><ul><li>Conserved genes having different functions </li></ul></ul>
  11. 11. Conserved genes, Similar functions <ul><li>Genes have: </li></ul><ul><ul><li>Homologous functions </li></ul></ul><ul><ul><li>Involved in the development of conserved structures in both humans and flies </li></ul></ul><ul><li>Mutations in both human </li></ul><ul><li>and fly homologues affect </li></ul><ul><li>same tissue/cell type </li></ul>
  12. 12. Defects in heart specification and function tinman NKX2-5 Malformations of mesodermal derivatives twist TWIST1 Defects of the auditory system salm or salr SALL1 Defects of the eyes eyeless PAX6 Alteration of anterior-posterior identities Hox genes Hox genes Affect when mutated Drosophila gene Human gene
  13. 13. Conserved genes, Similar functions <ul><li>Regulators of expression of effector genes </li></ul><ul><li>Sometimes effects on the transcription of target genes differ between fly and vertebrate </li></ul><ul><ul><li>Flies: twist activates FGFR (Fibroblast Growth Factor Receptor) </li></ul></ul><ul><ul><li>Mammals: TWIST1 negatively regulates Fgfr2 </li></ul></ul><ul><li>Hox genes differ in their detailed nature of target recognition </li></ul><ul><ul><li>Overall proteins function in a homologous manner to determine cell fate </li></ul></ul><ul><li>Recognition of DNA binding sites on target genes remains evolutionarily conserved </li></ul><ul><li>Enhancer sequence containing DNA binding site may have changed slightly due to natural selection </li></ul>
  14. 14. Conserved genes, Different functions <ul><li>Common signaling pathways </li></ul><ul><ul><li>Used several times in development </li></ul></ul><ul><ul><li>Also in species specific processes </li></ul></ul><ul><li>Notch pathway </li></ul><ul><ul><li>Homologous development function: </li></ul></ul><ul><ul><ul><li>Defines dorsal-ventral boundary of appendages in Drosophila </li></ul></ul></ul><ul><ul><ul><li>Establishes apical ectoderm ridge in vertebrate limbs </li></ul></ul></ul><ul><ul><ul><li>In both cases, regulated by glycosyl transferases in the Fringe family </li></ul></ul></ul><ul><ul><li>Species specific processes </li></ul></ul><ul><ul><ul><li>In vertebrates, essential for segmentation of somitic mesoderm and skeletal elements </li></ul></ul></ul><ul><ul><ul><li>In flies, limits the width of wing veins </li></ul></ul></ul><ul><ul><ul><li>Species specific structures </li></ul></ul></ul><ul><ul><ul><li>Relevant inferences can be drawn from one system to the other </li></ul></ul></ul>
  15. 15. Conserved genes, Different function <ul><li>Discovery of Delta in Drosophila </li></ul><ul><ul><li>Encodes a cell-surface ligand for the notch receptor </li></ul></ul><ul><ul><li>Mutated in Drosophila – thickens the wings </li></ul></ul><ul><ul><li>Loss of function in vertebrate homologue – related spinal malformations </li></ul></ul><ul><li>Served as a guide to discover other human homologues of Delta </li></ul><ul><ul><li>JAG1 (jagged1) and DLL3 (delta-like 3) </li></ul></ul><ul><ul><li>When mutated, see similar spinal abnormailites observed in human diseases </li></ul></ul><ul><ul><ul><li>Alagille syndrome and spondylocostal dysotosis </li></ul></ul></ul><ul><li>Advantage of fly model: </li></ul><ul><ul><li>Ability to identify and encourage further identification of genes associated with similar disease phenotypes </li></ul></ul>
  16. 16. Human disease: Neurological disorders <ul><li>Disorders that affect: </li></ul><ul><ul><li>Central nervous system (brain, brainstem and cerebellum) </li></ul></ul><ul><ul><li>Peripheral nervous system (Peripheral nerves – cranial nerves) </li></ul></ul><ul><ul><li>Autonomic nervous system (Parts of which are located both in the central and peripheral nervous systems) </li></ul></ul><ul><li>Four types currently studied in Drosophila: </li></ul><ul><ul><li>Triple-repeat diseases </li></ul></ul><ul><ul><li>Parkinson’s Disease </li></ul></ul><ul><ul><li>Familial Alzheimer disease </li></ul></ul><ul><ul><li>Fragile X syndrome </li></ul></ul>
  17. 17. Neurological disorders: Triple-repeat diseases <ul><li>Includes: </li></ul><ul><ul><li>Spinobulbar muscular atrophy </li></ul></ul><ul><ul><li>Spinal cerebellar ataxias </li></ul></ul><ul><ul><li>Huntington disease </li></ul></ul><ul><li>Extended consecutive repeat of a codon </li></ul><ul><ul><li>Glutamine encoding triplet CAG </li></ul></ul><ul><ul><li>Leads to neuronal degeneration </li></ul></ul><ul><ul><li>Longer repeats – earlier onset </li></ul></ul>
  18. 18. Neurological disorder: Triple-repeat diseases <ul><li>Mutant polyglutamine genes </li></ul><ul><ul><li>induce neuronal degeneration in fly retina </li></ul></ul><ul><ul><li>Mimics retinal degeneration in humans </li></ul></ul><ul><ul><li>Inclusion bodies present with extended CAG repeats </li></ul></ul><ul><li>Discovery of other genes involved in retinal degeneration </li></ul><ul><ul><li>Heat-shock proteins – chaperonins that re-fold misfolded proteins </li></ul></ul><ul><ul><li>protein degradation genes </li></ul></ul><ul><ul><li>histone deacetylation genes </li></ul></ul><ul><ul><li>apoptotic genes </li></ul></ul><ul><ul><li>genes encoding RNA binding proteins </li></ul></ul>
  19. 19. Neurological disorder: Triple-repeat diseases <ul><li>Some of these genes may regulate/clear inclusion bodies </li></ul><ul><ul><li>Expression of HSP70 in vertebrates </li></ul></ul><ul><ul><li>Expression of histone deacetlyase inhibitors in mice </li></ul></ul><ul><ul><li>Reduce effects of overexpressing expanded polyglutamine proteins. </li></ul></ul><ul><li>Advantage of fly model: </li></ul><ul><ul><li>Can validate activity of small molecule candidates to be used as therapeutic agents </li></ul></ul>
  20. 20. Neurological disorders: Parkinson’s Disease <ul><li>Progressive loss of dopaminergic neurons in the brainstem </li></ul><ul><li>Commonly studied human gene SNCA </li></ul><ul><ul><li>Encodes α -synuclein protein </li></ul></ul><ul><ul><li>Present in presynaptic terminals </li></ul></ul><ul><ul><li>Formation of Lewy bodies (cytoplasmic aggregate) </li></ul></ul><ul><ul><li>No obvious fly homologue </li></ul></ul><ul><li>Misexpression of mutant human gene in flies leads to late onset neurodegeneration in the eye </li></ul><ul><li>Flies have lead to discovery of additional genes which interact with α-synuclein </li></ul><ul><ul><li>Overlaps with those involved in polyglutamine disorders </li></ul></ul><ul><ul><li>Includes distinct set of genes </li></ul></ul>
  21. 21. Neurological disorders: Parkinson’s Disease <ul><li>Ubiquitin pathway </li></ul><ul><ul><li>accumulation of α-synuclein </li></ul></ul><ul><li>Parkinson’s Disease caused by mutations in PARK2 gene </li></ul><ul><ul><li>Encodes parkin, an e3-ligase </li></ul></ul><ul><ul><ul><li>Attaches ubiquitin to lysines of proteins to be destroyed </li></ul></ul></ul><ul><ul><li>When not mutated, forms a complex with α-synuclein </li></ul></ul><ul><ul><li>Mutation of fly homologue, park: </li></ul></ul><ul><ul><li>Degenerates flight muscles </li></ul></ul><ul><ul><li>Makes the fly more sensitive to free radicals </li></ul></ul><ul><ul><ul><li>Similar to sensitivity of dopaminergic neurons to toxin induced degeneration </li></ul></ul></ul><ul><li>Overexpression of park rescues effects of α-synuclein in the eye </li></ul>
  22. 22. Neurological Disorders: Familial Alzheimer disease (FAD) <ul><li>Responsible genes well-studied in flies </li></ul><ul><ul><li>Presenilin genes </li></ul></ul><ul><ul><li>Transmembrane proteases </li></ul></ul><ul><ul><li>Cleaves β-amyloid ( APP ) </li></ul></ul><ul><ul><ul><li>Transmembrane protein in extracellular plaques found in brains of FAD patients </li></ul></ul></ul><ul><ul><li>Normal function of APP : </li></ul></ul><ul><ul><ul><li>Mediates cell-surface signaling </li></ul></ul></ul><ul><ul><ul><li>Functions as a receptor for kinesin-dependent transport of specific cargo molecules along axons </li></ul></ul></ul><ul><ul><ul><li>Binds Cu 2+ and reduces its neurotoxicity </li></ul></ul></ul>
  23. 23. Neurological Disorders: Familial Alzheimer disease (FAD) <ul><li>Mutations in human APP causes FAD </li></ul><ul><ul><li>Unclear which function, when disrupted, is the one responsible for development of FAD </li></ul></ul><ul><li>Mutant Presenilin genes lead to accumulation of APP proteins in plaques </li></ul><ul><li>Drosophila homologue of APP ( Appl ) leads to premature death when mutated </li></ul>
  24. 24. Neurological disorders: Fragile X syndrome <ul><li>Mental retardation, associated with autism </li></ul><ul><li>Expansion of non-coding CGG repeat </li></ul><ul><li>Loss of function FMR1 (Fragile X mental retardation 1) gene </li></ul><ul><ul><li>RNA binding protein </li></ul></ul><ul><ul><li>Negatively regulates translation of: </li></ul></ul><ul><ul><ul><li>Genes that function at synapses for normal dendrite morphology </li></ul></ul></ul><ul><li>Mutant triple-repeat gene </li></ul><ul><ul><li>Heterozygous carriers </li></ul></ul><ul><ul><ul><li>Neuronal degeneration </li></ul></ul></ul><ul><ul><li>Homozygous carriers </li></ul></ul><ul><ul><ul><li>Do not express FMR1 and suffer no neuronal degeneration, only mental retardation </li></ul></ul></ul>
  25. 25. Neurological Disorders: Fragile X syndrome <ul><li>In the fly eye: </li></ul><ul><ul><li>Expanded CGG causes neurodegeneration </li></ul></ul><ul><ul><li>Wildtype CGG numbers do not </li></ul></ul><ul><li>Overexpression of other non-coding triplet, CAG also leads to neurodegenration </li></ul><ul><ul><li>Suppressed by HSP70 </li></ul></ul><ul><ul><li>Therefore triplet RNAs associated with aggregates acted upon by HSP70 </li></ul></ul><ul><li>As non-coding, neural degeneration phenotype could be mediated exclusively at RNA level </li></ul>
  26. 26. Human disease: Cancer <ul><li>Abnormal growth of cells </li></ul><ul><li>Cancer in Drosophila </li></ul><ul><ul><li>Short lived organism </li></ul></ul><ul><ul><li>Therefore does not naturally develop cancer manifested by lethal tumour overgrowth and metastasis </li></ul></ul><ul><li>Genes that affect cell cycle control and epithelial integrity recovered and studied </li></ul><ul><ul><li>Homolgous genes have important roles in formation and dispersion of tumours in humans </li></ul></ul>
  27. 27. Cancer: RTK-RAS-MAPK signaling <ul><li>RTK - receptor tyrosine kinase </li></ul><ul><li>RAS - proteins that bind GDP and release GTP as a second messenger </li></ul><ul><li>MAPK - mitogen-activated protein kinase </li></ul><ul><ul><li>Serine/threonine-specific protein kinase </li></ul></ul><ul><ul><li>Responds to extracellular stimuli (mitogens) </li></ul></ul><ul><ul><li>Regulates various cellular activities </li></ul></ul><ul><ul><ul><li>Gene expression </li></ul></ul></ul><ul><ul><ul><li>Mitosis </li></ul></ul></ul><ul><ul><ul><li>Differentiation </li></ul></ul></ul><ul><ul><ul><li>Cell survival/apoptosis </li></ul></ul></ul>RAS MAPK Mitogen RTK GDP GTP
  28. 28. Cancer: RTK-RAS-MAPK signaling <ul><li>First use of Drosophila to address cancer </li></ul><ul><ul><li>Construction of general pathway </li></ul></ul><ul><ul><li>Link between biochemical component and gene hierarchy </li></ul></ul><ul><ul><ul><li>Connected cell-surface receptors to internal regulation of target genes </li></ul></ul></ul><ul><li>Lead to discovery of specific genes in specific pathways and their interactions </li></ul><ul><ul><li>Wingless </li></ul></ul><ul><ul><li>Hedgehog </li></ul></ul><ul><ul><li>TGF- β </li></ul></ul><ul><ul><li>Notch </li></ul></ul><ul><li>All implemented in human cancer </li></ul>
  29. 29. Cancer: Target of Rapamycin (TOR) pathway <ul><li>Excessive cell growth </li></ul><ul><ul><li>Formation of benign tumours, such as in Tuberous sclerosis </li></ul></ul><ul><li>Mutations in TSC1 or TSC2 </li></ul><ul><ul><li>Form complex and act as GTPase protein </li></ul></ul><ul><ul><li>Inactivate RAS protein: RAS homologue enriched in brain (RHEB) </li></ul></ul><ul><ul><ul><li>RHEB enhances TOR signaling </li></ul></ul></ul><ul><ul><ul><ul><li>Enahnces protein synthesis </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Inhibits autophagy </li></ul></ul></ul></ul><ul><ul><li>Insulin pathway inactivates TSC1/2, thus activating TOR signaling </li></ul></ul><ul><ul><li>PTEN inactivates insulin signaling, thus activation TSC1/2 and inactivating TOR </li></ul></ul><ul><ul><ul><li>Mutations in PTEN activate insulin signaling, thus TOR </li></ul></ul></ul><ul><ul><ul><li>Leads to excess cell growth </li></ul></ul></ul>
  30. 30. PI3K Akt Tsc1 Tsc2 RHEB TOR PTEN Protein synthesis And cellular growth Insulin
  31. 31. Cancer: Cell cycle control <ul><li>Cancer sometimes caused by disruption of components at check points </li></ul><ul><ul><li>Negatively regulate cell cycle under normal conditions </li></ul></ul><ul><li>Drosophila homologues: </li></ul><ul><ul><li>Cyclins, cyclin dependent kinases, E2F genes (enhance cell cycle progression) </li></ul></ul><ul><ul><li>CDKN2B (decapo), C1B1 (kip) and retinoblastoma protein (Rb) (inhibit cell cycle progression) </li></ul></ul><ul><ul><li>P53 –downstream, pro-apoptotic effector of E2F genes </li></ul></ul><ul><li>Flies have one copy of these genes </li></ul><ul><ul><li>Vertebrates often have several </li></ul></ul>
  32. 32. Cancer: Cell Cycle Control <ul><li>Searching for tumour suppressors in Drosophila leading to cellular growth (like PTEN) </li></ul><ul><ul><li>Discovered previously unknown negative regulators of cell cycle: </li></ul></ul><ul><ul><ul><li>Warts ( WTS or LATS ) </li></ul></ul></ul><ul><ul><ul><li>Salvadore ( SAV ) </li></ul></ul></ul><ul><ul><ul><li>Hippo </li></ul></ul></ul><ul><li>Motivated studies in mice and humans to confirm importance of new genes in tumourgenesis </li></ul><ul><ul><li>LATS1 mutant mice – tumour overgrowths (like fly) </li></ul></ul><ul><ul><li>Human renal and colon cancer cell lines – mutations in SAV homologue </li></ul></ul><ul><li>Flies help clarify cell cycle control mechanisms and lead to identification of new genes which may prevent excessive cell proliferation and cancer </li></ul>
  33. 33. Cancer: Tumour metastasis <ul><li>Not observed in wild-type flies </li></ul><ul><li>Instead, study genes involved in regulation of cell behaviours </li></ul><ul><ul><li>Migration </li></ul></ul><ul><ul><li>Invasion of epithelial sheets </li></ul></ul><ul><li>Show mechanistic similarities to processes involved in multistep spread of cancer cells </li></ul><ul><li>Normal cells can undergo programmed migrations, and then invasion of epithelial sheet </li></ul><ul><ul><li>Two distinct steps </li></ul></ul>
  34. 34. Cancer: Tumour metastasis <ul><li>Screen to find genes involved in metastasis </li></ul><ul><ul><li>Identified mutations in scribbled ( scrib ) </li></ul></ul><ul><ul><ul><li>Maintains normal apical/basal cell polarity </li></ul></ul></ul><ul><ul><ul><li>Scrib mutants – overproliferation of cells </li></ul></ul></ul><ul><ul><li>When have both </li></ul></ul><ul><ul><ul><li>Mutated form of Drosophila RAS </li></ul></ul></ul><ul><ul><ul><li>A loss-of-function scrib </li></ul></ul></ul><ul><ul><li>Cells break free and move to other locations </li></ul></ul><ul><ul><li>Migration also seen with notch mutations combined with scrib mutants </li></ul></ul><ul><ul><li>Like mammalian tumours, E-cadherin is downregulated </li></ul></ul><ul><ul><ul><li>Adhesion molecule which would prevent metastasis </li></ul></ul></ul><ul><li>Invasive cancer thus results from distinct steps and separate processes which can be studied in Drosophila </li></ul>
  35. 35. Limitations of fly models <ul><li>Some biological processes evolved in vertebrate lineage only </li></ul><ul><ul><li>Genes involved in creating four-chambered heart </li></ul></ul><ul><ul><li>However, could study genes in specific steps of these processes </li></ul></ul><ul><li>Smaller organism, such as yeast, might be preferred when studying cell-autonomous functions (ie: DNA repair) </li></ul><ul><ul><li>Shorter generation time, smaller genome, large number of individuals produced </li></ul></ul><ul><li>Ideal study of human disease might be: </li></ul><ul><ul><li>Parallel analysis of gene at all relevant tiers </li></ul></ul><ul><ul><ul><li>Cell autonoumous effects in yeast </li></ul></ul></ul><ul><ul><ul><li>Multicellular or inductive events mediated by gene in Drosophila </li></ul></ul></ul><ul><ul><ul><li>Accurate disease model and mutations of gene in mice </li></ul></ul></ul>
  36. 36. Summary <ul><li>Benefits of Drosophila include: </li></ul><ul><ul><li>Broad spectrum of genes related to human disease already discovered </li></ul></ul><ul><ul><li>Many successful techniques already developed </li></ul></ul><ul><ul><li>Already a powerful tool in study of developmental and neurological disorders, and cancer </li></ul></ul><ul><li>Future Perpectives: </li></ul><ul><ul><li>Identification of novel genes functioning in disease processes </li></ul></ul><ul><ul><li>Determination of genes contributing to complex disorders </li></ul></ul><ul><ul><li>Exploit the fly to answer already existing questions, and formulate new hypotheses </li></ul></ul><ul><li>Drosophila is a most effective model: </li></ul><ul><li>More simplicity than vertebrate models </li></ul><ul><li>Greater complexity than yeast or bacteria models </li></ul>
  37. 37. Reference <ul><li>Bier, E. 2005. Drosophila, the Golden Bug, Emerges as a Tool for Human Genetics. Nature Reviews Genetics 6: 9-23 </li></ul>