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Jennifer Slade B.Sc (Hon), M.Sc Candidate

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

2 Outline Model organism Drosophila as a 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 Human gene Drosophila gene Affect when mutated Hox genes Alteration of anterior-posterior identities PAX6 eyeless Defects of the eyes SALL1 salm or salr Defects of the auditory system TWIST1 twist Malformations of mesodermal derivatives NKX2-5 tinman Defects in heart specification and function

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 Cu2+ 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 Mitogen RTK RAS GDP GTP MAPK

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

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: Future Perpectives:
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 Drosophila, the Golden Bug, Emerges as a Tool for Human Genetics. Nature Reviews Genetics 6: 9-23

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