Model Organisms

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

• Drosophila
• Xenopus
• Zebrafish
• Mouse
• C. elegans
• Yeast
• E. coli
• Arabidopsis
 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
Drosophila melanogaster
as a model organism
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
 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
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.
 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
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
Human gene Drosophila gene Affect when mutated

Hox genes 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
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
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
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
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
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
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
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
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
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
 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
 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
 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
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
 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
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 Mitogen

– Responds to extracellular stimuli (mitogens)

– Regulates various cellular activities RTK
• Gene expression RAS
• Mitosis
GDP GTP
• Differentiation
• Cell survival/apoptosis
MAPK
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
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
Insulin

PTEN
Akt

PI3K
Tsc1 Tsc2

Protein synthesis
And cellular growth

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