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Model Organisms

Model Organisms

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Model Organisms

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  1. Model Organisms Jennifer Slade B.Sc (Hon), M.Sc Candidate

  2. Model organism Definition Current models Characterisitics of a “good” model organism? Drosophila as a model organism Characteristics Uses in Research Developmental disorders Conserved genes, similar functions Conserved genes, different functions Neurological disorders Triple-repeat diseases Parkinson disease Familial Alzheimer disease Fragile X Cancer RTK-RAS-MAPK signaling Targets of Rapamycin pathway Cell cycle control Tumour metastasis Limitations of fly models Summary Outline

  3. Definition of Model Organism • Specific species or organism • Extensively studied in research laboratories • Advance our understanding of • Cellular function • Development • Disease • Ability to apply new knowledge to other organisms

  4. Current Models • Drosophila • Xenopus • Zebrafish • Mouse • C. elegans • Yeast • E. coli • Arabidopsis

  5. Characteristics of a “Good” Model Organism • Think individually • Make jot notes • 5 to 10 minutes • Share in groups • Get in groups of 4 • Discuss various characteristics • Share with the class • One person from each group write one characteristic discussed on board • Explain why characteristic is beneficial

  6. Drosophila melanogasteras a model organism

  7. Characteristics of Drosophila that make it a good model organism • Small, easy and cheap to maintain and manipulate • Short lifespan • Produce large numbers of offspring • Development is external • Availability of mutants • Lots of history/previous experiments and discoveries • Genome is sequenced • Homologues for at least 75 % of human disease genes • Exhibit complex behaviours • Fewer ethical concerns

  8. Drosophila in Research • Early research aided in the understanding of development • Made first link between chromosome and phenotype • Identified various genes and mechanisms of development • Current research focuses on the study of human disease • Developmental disorders • Neurological disorders • Cancer

  9. Technique: Second site modifier screen • Begin with a fly posessing a mutant phenotype • Create random mutations that might effect this phenotype in this genetic background • Via radiation or feeding of a mutagen • Observe offspring or “grandoffspring” for either less or much more severe phenotype • Some might be revertants of the original gene • Others might be mutants for upstream or downstream components of the pathway(s) that lead to the original phenotype • Rarely, there might be mutants of a gene with a compensational function. • These are second site mutants.

  10. Human Disease : Developmental Disorders • Dysmorphologies • Diseases resulting in morphological defects • Largest, most prevalent human genetic disorders • Result from mutations in genes that control important steps in development, such as: • Transcription factors • Proteins involved in signal transduction • Two broad categories: • Conserved genes with orthologous function • Conserved genes having different functions

  11. Conserved genes, Similar functions • Genes have: • Homologous functions • Involved in the development of conserved structures in both humans and flies • Mutations in both human and fly homologues affect same tissue/cell type

  12. Conserved genes, Similar functions • Regulators of expression of effector genes • Sometimes effects on the transcription of target genes differ between fly and vertebrate • Flies: twist activates FGFR (Fibroblast Growth Factor Receptor) • Mammals: TWIST1 negatively regulates Fgfr2 • Hox genes differ in their detailed nature of target recognition • Overall proteins function in a homologous manner to determine cell fate • Recognition of DNA binding sites on target genes remains evolutionarily conserved • Enhancer sequence containing DNA binding site may have changed slightly due to natural selection

  13. Conserved genes, Different functions • Common signaling pathways • Used several times in development • Also in species specific processes • Notch pathway • Homologous development function: • Defines dorsal-ventral boundary of appendages in Drosophila • Establishes apical ectoderm ridge in vertebrate limbs • In both cases, regulated by glycosyl transferases in the Fringe family • Species specific processes • In vertebrates, essential for segmentation of somitic mesoderm and skeletal elements • In flies, limits the width of wing veins • Species specific structures • Relevant inferences can be drawn from one system to the other

  14. Conserved genes, Different function • Discovery of Delta in Drosophila • Encodes a cell-surface ligand for the notch receptor • Mutated in Drosophila – thickens the wings • Loss of function in vertebrate homologue – related spinal malformations • Served as a guide to discover other human homologues of Delta • JAG1 (jagged1) and DLL3 (delta-like 3) • When mutated, see similar spinal abnormailites observed in human diseases • Alagille syndrome and spondylocostal dysotosis • Advantage of fly model: • Ability to identify and encourage further identification of genes associated with similar disease phenotypes

  15. Human disease: Neurological disorders • Disorders that affect: • Central nervous system (brain, brainstem and cerebellum) • Peripheral nervous system (Peripheral nerves – cranial nerves) • Autonomic nervous system (Parts of which are located both in the central and peripheral nervous systems) • Four types currently studied in Drosophila: • Triple-repeat diseases • Parkinson’s Disease • Familial Alzheimer disease • Fragile X syndrome

  16. Neurological disorders: Triple-repeat diseases • Includes: • Spinobulbar muscular atrophy • Spinal cerebellar ataxias • Huntington disease • Extended consecutive repeat of a codon • Glutamine encoding triplet CAG • Leads to neuronal degeneration • Longer repeats – earlier onset

  17. Neurological disorder: Triple-repeat diseases • Mutant polyglutamine genes • induce neuronal degeneration in fly retina • Mimics retinal degeneration in humans • Inclusion bodies present with extended CAG repeats • Discovery of other genes involved in retinal degeneration • Heat-shock proteins – chaperonins that re-fold misfolded proteins • protein degradation genes • histone deacetylation genes • apoptotic genes • genes encoding RNA binding proteins

  18. Neurological disorder: Triple-repeat diseases • Some of these genes may regulate/clear inclusion bodies • Expression of HSP70 in vertebrates • Expression of histone deacetlyase inhibitors in mice • Reduce effects of overexpressing expanded polyglutamine proteins. • Advantage of fly model: • Can validate activity of small molecule candidates to be used as therapeutic agents

  19. Neurological disorders: Parkinson’s Disease • Progressive loss of dopaminergic neurons in the brainstem • Commonly studied human gene SNCA • Encodes α-synuclein protein • Present in presynaptic terminals • Formation of Lewy bodies (cytoplasmic aggregate) • No obvious fly homologue • Misexpression of mutant human gene in flies leads to late onset neurodegeneration in the eye • Flies have lead to discovery of additional genes which interact with α-synuclein • Overlaps with those involved in polyglutamine disorders • Includes distinct set of genes

  20. Neurological disorders: Parkinson’s Disease • Ubiquitin pathway • accumulation of α-synuclein • Parkinson’s Disease caused by mutations in PARK2 gene • Encodes parkin, an e3-ligase • Attaches ubiquitin to lysines of proteins to be destroyed • When not mutated, forms a complex with α-synuclein • Mutation of fly homologue, park: • Degenerates flight muscles • Makes the fly more sensitive to free radicals • Similar to sensitivity of dopaminergic neurons to toxin induced degeneration • Overexpression of park rescues effects of α-synuclein in the eye

  21. Neurological Disorders: Familial Alzheimer disease (FAD) • Responsible genes well-studied in flies • Presenilin genes • Transmembrane proteases • Cleaves β-amyloid (APP) • Transmembrane protein in extracellular plaques found in brains of FAD patients • Normal function of APP: • Mediates cell-surface signaling • Functions as a receptor for kinesin-dependent transport of specific cargo molecules along axons • Binds Cu2+ and reduces its neurotoxicity

  22. Neurological Disorders: Familial Alzheimer disease (FAD) • Mutations in human APP causes FAD • Unclear which function, when disrupted, is the one responsible for development of FAD • Mutant Presenilin genes lead to accumulation of APP proteins in plaques • Drosophila homologue of APP (Appl) leads to premature death when mutated

  23. Neurological disorders: Fragile X syndrome • Mental retardation, associated with autism • Expansion of non-coding CGG repeat • Loss of function FMR1 (Fragile X mental retardation 1) gene • RNA binding protein • Negatively regulates translation of: • Genes that function at synapses for normal dendrite morphology • Mutant triple-repeat gene • Heterozygous carriers • Neuronal degeneration • Homozygous carriers • Do not express FMR1 and suffer no neuronal degeneration, only mental retardation

  24. Neurological Disorders: Fragile X syndrome • In the fly eye: • Expanded CGG causes neurodegeneration • Wildtype CGG numbers do not • Overexpression of other non-coding triplet, CAG also leads to neurodegenration • Suppressed by HSP70 • Therefore triplet RNAs associated with aggregates acted upon by HSP70 • As non-coding, neural degeneration phenotype could be mediated exclusively at RNA level

  25. Human disease: Cancer • Abnormal growth of cells • Cancer in Drosophila • Short lived organism • Therefore does not naturally develop cancer manifested by lethal tumour overgrowth and metastasis • Genes that affect cell cycle control and epithelial integrity recovered and studied • Homolgous genes have important roles in formation and dispersion of tumours in humans

  26. Cancer: RTK-RAS-MAPK signaling • RTK - receptor tyrosine kinase • RAS - proteins that bind GDP and release GTP as a second messenger • MAPK - mitogen-activated protein kinase • Serine/threonine-specific protein kinase • Responds to extracellular stimuli (mitogens) • Regulates various cellular activities • Gene expression • Mitosis • Differentiation • Cell survival/apoptosis Mitogen RTK RAS GDP GTP MAPK

  27. Cancer: RTK-RAS-MAPK signaling • First use of Drosophila to address cancer • Construction of general pathway • Link between biochemical component and gene hierarchy • Connected cell-surface receptors to internal regulation of target genes • Lead to discovery of specific genes in specific pathways and their interactions • Wingless • Hedgehog • TGF-β • Notch • All implemented in human cancer

  28. Cancer: Target of Rapamycin (TOR) pathway • Excessive cell growth • Formation of benign tumours, such as in Tuberous sclerosis • Mutations in TSC1 or TSC2 • Form complex and act as GTPase protein • Inactivate RAS protein: RAS homologue enriched in brain (RHEB) • RHEB enhances TOR signaling • Enahnces protein synthesis • Inhibits autophagy • Insulin pathway inactivates TSC1/2, thus activating TOR signaling • PTEN inactivates insulin signaling, thus activation TSC1/2 and inactivating TOR • Mutations in PTEN activate insulin signaling, thus TOR • Leads to excess cell growth

  29. Insulin PTEN Akt PI3K Tsc1 Tsc2 Protein synthesis And cellular growth RHEB TOR

  30. Cancer: Cell cycle control • Cancer sometimes caused by disruption of components at check points • Negatively regulate cell cycle under normal conditions • Drosophila homologues: • Cyclins, cyclin dependent kinases, E2F genes (enhance cell cycle progression) • CDKN2B (decapo), C1B1 (kip) and retinoblastoma protein (Rb) (inhibit cell cycle progression) • P53 –downstream, pro-apoptotic effector of E2F genes • Flies have one copy of these genes • Vertebrates often have several

  31. Cancer: Cell Cycle Control • Searching for tumour suppressors in Drosophila leading to cellular growth (like PTEN) • Discovered previously unknown negative regulators of cell cycle: • Warts (WTS or LATS) • Salvadore (SAV) • Hippo • Motivated studies in mice and humans to confirm importance of new genes in tumourgenesis • LATS1 mutant mice – tumour overgrowths (like fly) • Human renal and colon cancer cell lines – mutations in SAV homologue • Flies help clarify cell cycle control mechanisms and lead to identification of new genes which may prevent excessive cell proliferation and cancer

  32. Cancer: Tumour metastasis • Not observed in wild-type flies • Instead, study genes involved in regulation of cell behaviours • Migration • Invasion of epithelial sheets • Show mechanistic similarities to processes involved in multistep spread of cancer cells • Normal cells can undergo programmed migrations, and then invasion of epithelial sheet • Two distinct steps

  33. Cancer: Tumour metastasis • Screen to find genes involved in metastasis • Identified mutations in scribbled (scrib) • Maintains normal apical/basal cell polarity • Scrib mutants – overproliferation of cells • When have both • Mutated form of Drosophila RAS • A loss-of-function scrib • Cells break free and move to other locations • Migration also seen with notch mutations combined with scrib mutants • Like mammalian tumours, E-cadherin is downregulated • Adhesion molecule which would prevent metastasis • Invasive cancer thus results from distinct steps and separate processes which can be studied in Drosophila

  34. Limitations of fly models • Some biological processes evolved in vertebrate lineage only • Genes involved in creating four-chambered heart • However, could study genes in specific steps of these processes • Smaller organism, such as yeast, might be preferred when studying cell-autonomous functions (ie: DNA repair) • Shorter generation time, smaller genome, large number of individuals produced • Ideal study of human disease might be: • Parallel analysis of gene at all relevant tiers • Cell autonoumous effects in yeast • Multicellular or inductive events mediated by gene in Drosophila • Accurate disease model and mutations of gene in mice

  35. Summary • Benefits of Drosophila include: • Broad spectrum of genes related to human disease already discovered • Many successful techniques already developed • Already a powerful tool in study of developmental and neurological disorders, and cancer • Future Perpectives: • Identification of novel genes functioning in disease processes • Determination of genes contributing to complex disorders • Exploit the fly to answer already existing questions, and formulate new hypotheses • Drosophila is a most effective model: • More simplicity than vertebrate models • Greater complexity than yeast or bacteria models

  36. Reference • Bier, E. 2005. Drosophila, the Golden Bug, Emerges as a Tool for Human Genetics. Nature Reviews Genetics 6: 9-23