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  • 1. Model Organisms Jennifer Slade B.Sc (Hon), M.Sc Candidate
  • 2. Outline
    • 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
  • 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 melanogaster as 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. 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. 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
  • 14. 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
  • 15. 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
  • 16. 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
  • 17. 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
  • 18. 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
  • 19. 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
  • 20. 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
  • 21. 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
  • 22. 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 Cu 2+ and reduces its neurotoxicity
  • 23. 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
  • 24. 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
  • 25. 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
  • 26. 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
  • 27. 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
    RAS MAPK Mitogen RTK GDP GTP
  • 28. 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
  • 29. 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
  • 30. PI3K Akt Tsc1 Tsc2 RHEB TOR PTEN Protein synthesis And cellular growth Insulin
  • 31. 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
  • 32. 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
  • 33. 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
  • 34. 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
  • 35. 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
  • 36. 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
  • 37. Reference
    • Bier, E. 2005. Drosophila, the Golden Bug, Emerges as a Tool for Human Genetics. Nature Reviews Genetics 6: 9-23