Models: NonMammalian

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Models Non-Mammalian

• Pamela J. Lein, Ph.D.
• Center for Research on Occupational and
  Environmental Toxicology
• Oregon Health Sciences University

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Collins et al., 2008, Science 319906-907.
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Why consider non-mammalian models for DNT?

• A major challenge with in vitro models for DNT is
  that in the developing nervous system the whole
  is greater than the sum of its parts
• Reducing developing nervous system to subsets of
  cell or slices of specific brain regions
  increases chance of missing critical differences
  and significant interactions between
• cell types
• brain regions
• organ systems
• developmental stages

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Scientific justification for considering
non-mammalian models for DNT

• The fish is a frogis a chickenis a mouse
  Charles Kimmel (1989)
  Trends Genet 5 283-288
• Fundamental mechanisms of neurodevelopment are
  remarkably conserved across species
• Simple organisms exhibit the same
  neurodevelopmental
• processes as humans
• Genes homologous to human genes implicated in
• neurodevelopment and neurodevelopmental
  diseases
• have been identified in simple organisms

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Logistic advantages of non-mammalian models
relative to mammalian models

• Small size, rapid embryonic development, and
  short life cycle
• Decreases costs
• Increases throughput
• Relative ease of transgenesis
• Facilitates mechanistic studies
• Provides powerful tools for optimizing model for
  DNT (increasing resolution, humanizing model)

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Non-mammalian models used widely to study
mechanisms of neurodevelopment
Nematodes (Caenorhabditis elegans) Sea
urchin Insects (Drosophila melanogaster) Fish
(Danio rerio) Amphibian (frog) Birds (chick
embyro) Mammals (mice, rats, non-human primates)
increasing complexity
Entire genomic sequence is known
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What kinds of endpoints can be assessed in
non-mammalian models?

• Gene expression
• Toxicity pathways
• Neurodevelopmental processes
• Behavior
• Multiple endpoints can be measured, across
  several organ systems (dev tox)

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Neurodevelopmental processes that occur in
nematodes, flies, zebrafish

No myelination in nematode and fly
Schematic courtesy of Bill Mundy, U.S.E.P.A.
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C. elegans as a model for DNT

• Simple nervous system
• 302 neurons
• Cell fate and neural circuits completely mapped

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Behavioral analyses of C. elegans

• Mechanosensory behavior
• Reproductive behavior
• Learning, adaptation and habituation

A. Sinusoidal forward locomotion B. Omega turn in
navigation C. Egg laying D. Solitary feeding
behavior E. Social feeding behavior Schafer,
2005, Current Biology 15R723-R729.
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DNT studies in C. elegansCole et al., 2004,
TAAP, 194248-256.
EC50 values in C. elegans determined using
computerized tracking of locomotion.
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Drosophila as a model for DNT

• Powerful tool for elucidating molecular
  mechanisms of neurodevelopment
• Easy to manipulate gene expression
• Being developed as a model for examining genes
  that contribute to the pathogenesis of
  neurological diseases (e.g., autism)
• Exhibit relatively complex behaviors

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Drosophila courtship a social interaction
modified by experience
A. Orienting
B. Tapping
C. Singing
partner selection communication
cooperation behavioral sequence
single-gene effects courtship
conditioning
D. Licking E. Attempted F.
Copulation Copulation
What is it about no you dont understand?
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DNT studies in Drosophila
?

• Few articles in the peer-reviewed literature
• Primarily mechanistic studies
• Heavy metals
• Organophosphorus anticholinesterases

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Zebrafish (Danio rerio) as DNT Model

• Key biological traits more similar to human than
  nematodes or flies and thus may make the
  zebrafish more immediately useful for DNT
• mechanisms of gene regulation differ
  significantly between invertebrates and
  vertebrates
• overall organization of the major components of
  the fish brain is highly homologous to the human
  brain
• zebrafish possesses all the classical sense
  modalities (vision, olfaction, taste, touch,
  balance, and hearing) and their sensory pathways
  share overall homology with humans.
• lesions of the structural homolog of the
  hippocampus in fish selectively impair spatial
  memory

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Major logistic advantage of zebrafish relative to
nematodes and flies

• Zebrafish embryos, which develop externally, are
  optically transparent

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Imaging of neurodevelopment in zebrafish

• possible to resolve individual cells in vivo
• across a broad range of developmental stages
• potential to use non-invasive methods to
• monitor neurodevelopment at different time
• points in the same animal
• Show movies

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Behavioral analyses in zebrafish
adapted from Elwood Linney, Duke University
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Strategy for evaluating effects of early
exposures on adult behavioradapted from Elwood
Linney, Duke University
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DNT studies in zebrafish

• Hill et al., 2005, Tox Sci 86 6-19
• Metals, PCBs/PAHs, retinoic acid, cyclopamine
  (hedgehog signaling), fragrances, nitrated
  benzenes, pesticides and herbicides, estrogenic
  compounds, nicotine, ethanol, others
• Ton et al., 2006, Birth Defects Res 76 553-567

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Challenges of adapting non-mammalian models for
DNT

• Technical challenges
• imaging (non-invasive, automation/high
  throughput)
• Toxicokinetics
• administration of chemicals
• determining dose
• metabolism
• Predictability
• is the response observed in non-mammalian models
  predictive of neurotoxic responses in the
  developing human nervous system?