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
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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
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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.
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 anteriorposterior 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:
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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
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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
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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
– Responds to extracellular stimuli (mitogens)
– Regulates various cellular activities
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Gene expression
Mitosis
Differentiation
Cell survival/apoptosis
Mitogen
RTK
RAS
GDP
MAPK
GTP
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
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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