Seeing the Elegance in C' elegans

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Seeing the Elegance in C. elegans

• Dr. Annette M. Parrott
• GIFT 2006

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What is Caenorhabditis elegans?

• Free-living soil nematode
• 3 day life cycle
• 1.5mm long adult
• 959 cells total 302 neurons

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Why study C. elegans?

• About 35 of C. elegans genes have human
  homologues

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C. elegans as a model organism

• Model organisms are easily manipulated and
  studied in the laboratory and lend scientific
  insights to other organisms.
• C. elegans was first studied as a model organism
  in 1965 by Sydney Brenner and its genome
  sequenced in 1998.

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Kaletta et al. Nature Reviews Drug Discovery
advance online publication published online 21
April 2006 doi10.1038/nrd2031
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My Research...

• I primarily investigated whether the oxygen
  concentration preference for C. elegans is a
  learned or innate behavior. 
• I also recorded which neurons and combinations of
  neurons (URX, AQR, PQR) were marked (with GFP) in
  KyIs342

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• Oxygen Sensation and Oxygen-Dependent Behavior
• Levels of oxygen vary widely in natural soil and
  water environments. C. elegans has rapid
  behavioral responses to oxygen that can be
  monitored when animals move through a gas-phase
  oxygen gradient. Wild-type C. elegans accumulate
  at 510 O2, avoiding both high and low oxygen
  levels.
• The standard laboratory C. elegans strain does
  not avoid high oxygen in the presence of food.
  Aerotaxis modulation by food is accomplished by
  npr-1 neuropeptide signaling, a TGFß
  (transforming growth factor-ß) peptide that
  regulates transcription, and the neurotransmitter
  serotonin. These interacting systems regulate a
  distributed network of oxygen-sensing neurons to
  mediate the switch from weak aerotaxis on food to
  strong aerotaxis in its absence. The complete
  neuroanatomical "wiring diagram" of the C.
  elegans nervous system does not include these
  regulatory pathways, indicating that functional
  experiments are needed to uncover circuits even
  in anatomically well-described nervous systems.
• Human applications include memory, learning,
  carotid body (hyperventilation), heathy blood
  pressure, depression, neural retworking

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• Current Research
• Aerotaxis results for gcy-35 rescuing strains and
  for tax-2 and tax-4 mutants, and oxygen-regulated
  behaviors in tax-4 mutants and double mutants.
• Aerotaxis assays were scored in nine bins of
  equal size that spanned the linear gradient from
  0-21 oxygen. To generate a hyperoxia avoidance
  index, we compared the distribution of animals
  between medium oxygen (bins 5-7, 4.66-11.67) and
  high oxygen (bins 1-4, 11.67-21), correcting for
  the smaller total area covered by bins 5-7 by
  calculating the average fraction of animals in
  each bin.
• Wild-type animals exhibited no preference in the
  absence of an oxygen gradient (N2 air control).

Hyperoxia B - A Avoidance
Index BA Hypoxia B -
C Avoidance Index BC
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Experimental Design

• 4L4s KyIs342 were plated to NGM and incubated at
  21 and 10 O2
• Air was refreshed daily
• (KyIs342 Isp gcy-32tax-4gfp)

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• On day 4, when there were ample adults, an oxygen
  gradient behavior assay was performed for
  approximately 25 minutes.

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• A gas-phase PDMS aerotaxis device, was used.
• A gradient was established by diffusion along the
  long axis of the device.

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• Measured oxygen concentrations in aerotaxis
  device (side view).

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Challenges

• Flooding of device
• Oxygen aggregation

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Results
Literature
My results
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Neuroscience

• Worms can sense heat, feel touches, smell, and
  taste (they cant hear or see)
• Neurons organize into layers and form
  circuits
• They compute information from the environment
• They tell the worm how to respond (e.g. flee the
  danger, or move towards food)
• Learning and memory

PQR
AQR URX
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Data
308 L1-L3 Surveyed
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Is there C. elegans beyond ChBE ???
OF COURSE there is!!!
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In the classroom...

• Alien World Observations 1st week (6 days, 1st
  15 minutes of class) of school (Biology Gifted
  Biology). Students spend several days making
  observations, as 1 worm becomes thousands of
  worms, reach carrying capacity and eventually
  start dying.
• Biomimetics Research 1st month of school
  (Gifted Biology). Students research applications
  of biological systems in technology we use daily.
• Genetics of C. elegans 1 week in November
  (AP Biology) Students predict and investigate
  inheritance in C. elegans mutants.
• Blast those Genes 1 day in October
  (Gifted, AP Biology). Students use worldwide
  genomic databases to find homologs between C.
  elegans and H. sapiens, and hypothesize the
  significance of such.
• Field Collected Nematodes Dichotomous Key 1
  week in January (Biology Gifted Biology).
  Students will field collect nematodes and compare
  to C. elegans
• Helpful/Harmful Animal Book 1st month, 2nd
  semester. (All Biology) Students will investigate
  helpful and harmful aspects of the 8 major animal
  phyla

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On the Web...
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In the journals...
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Summer 2007 ???
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Future ???
Not even the sky is the limit!
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References

• Brenner, S. The genetics of Caenorhabditis
  elegans. Genetics 77, 71-94 (1974).
• Gray JM, Karow DS, Lu H, Chang AJ, Chang JS,
  Ellis RE, Marletta MA, Bargmann CI. Oxygen
  sensation and social feeding mediated by a C.
  elegans guanylate cyclase homologue Nature 430,
  317-322 (15 July 2004)
• Kaletta, Titus and Michael O. Hengartner. Finding
  Function in Novel Targets. C. elegans as a Model
  Organism Nature Reviews Drug Discovery 5,
  387-399 (May 2006)
• Yarris, Lynn. Oxygen Sensing in Worms May Hold
  Key to Healthy Blood Pressure in Humans. July 14,
  2004. http//www.lbl.gov/Science-Articles/Archive/
  PBD-nematodes.html

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• Aerotaxis results for gcy-35 rescuing strains and
  for tax-2 and tax-4 mutants, and oxygen-regulated
  behaviors in tax-4 mutants and double mutants.
• Supplemental Fig. 2 Statistical analysis of
  aerotaxis data from Fig. 1, Suppl. Fig. 3, and
  other experiments. Aerotaxis assays were scored
  in nine bins of equal size that spanned the
  linear gradient from 0-21 oxygen. To generate a
  hyperoxia avoidance index, we compared the
  distribution of animals between medium oxygen
  (bins 5-7, 4.66-11.67) and high oxygen (bins
  1-4, 11.67-21), correcting for the smaller total
  area covered by bins 5-7 by calculating the
  average fraction of animals in each bin. A
  hyperoxia avoidance index was calculated as
  (Medium-High)/(MediumHigh) hyperoxia avoidance
  of 1.0 represents complete exclusion of animals
  from bins 1-4 in the aerotaxis assay, 0.0
  represents indifference to hyperoxia and 1.0
  represents complete exclusion from bins 5-7.
  Wild-type animals exhibited no preference in the
  absence of an oxygen gradient (N2 air control).
  Results were analyzed by ANOVA and Bonferroni
  t-test. indicates strains or conditions that
  were significantly defective in avoidance of
  hyperoxia compared to N2 in the absence of food
  (plt0.05). indicates transgenic gcy-35 strains
  that were significantly rescued compared to
  gcy-35(ok769) (plt0.05). The gcy-35 defect was
  fully rescued by the gcy-35 cDNA expressed in
  URX, AQR, and PQR under the gcy-32 promoter (2
  lines), and was partially rescued by a
  gcy-36gcy-35 clone (1 line) and a gcy-35
  genomic clone (1 line). The tax-4(ks28) defect
  was fully rescued by tax-4 expressed in URX, AQR
  and PQR under the gcy-32 promoter7. N2 animals
  on food were highly defective in hyperoxia
  avoidance (), whereas npr-1(g320) and
  npr-1(ad609) on food had normal or enhanced
  hyperoxia avoidance ().

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• Oxygen Sensation and Oxygen-Dependent
  BehaviorLevels of oxygen vary widely in natural
  soil and water environments. C. elegans has rapid
  behavioral responses to oxygen that can be
  monitored when animals move through a gas-phase
  oxygen gradient. Wild-type C. elegans accumulates
  at about 510 percent oxygen, avoiding both high
  and low oxygen levels. Normal aerotaxis behavior
  requires at least three different soluble
  guanylate cyclases (sGCs) that can directly bind
  oxygen through their heme domains (work of our
  collaborators, David Karow and Michael Marletta
  University of California, Berkeley). These
  results suggest that sGCs are a new class of
  molecular receptor for environmental oxygen.
• Oxygen levels can modify behavioral responses to
  other stimuli. One behavior that is regulated by
  oxygen is aggregation, or social feeding.
  Aggregation is rare in the standard laboratory
  strain of C. elegans but prominent in wild C.
  elegans isolates that bear the neuropeptide
  receptor allele npr-1(215F). Aggregation in
  npr-1(215F) strains occurs only at high oxygen
  levels and is controlled by sensory neurons that
  detect oxygen. Oxygen levels are low in
  aggregates, so aggregation appears to be a
  strategy for avoiding hyperoxia.
• The standard laboratory C. elegans strain does
  not avoid high oxygen in the presence of food.
  Aerotaxis modulation by food is accomplished by
  npr-1 neuropeptide signaling, a TGFß
  (transforming growth factor-ß) peptide that
  regulates transcription, and the neurotransmitter
  serotonin. These interacting systems regulate a
  distributed network of oxygen-sensing neurons to
  mediate the switch from weak aerotaxis on food to
  strong aerotaxis in its absence. The complete
  neuroanatomical "wiring diagram" of the C.
  elegans nervous system does not include these
  regulatory pathways, indicating that functional
  experiments are needed to uncover circuits even
  in anatomically well-described nervous systems.
• The discovery of the oxygen-sensing mechanism in
  nematodes actually began as an investigation into
  a human enzyme, guanylate cyclase, which performs
  signaling interactions with nitric oxide
  molecules critical to regulating blood pressure.
  When nitric oxide enters a human cell it
  activates guanylate cyclase, which catalyzes the
  formation of cyclic GMP, a protein that relaxes
  and dilates blood vessels. The ability of
  guanylate cyclase to sense and interact with
  nitric oxide also plays a prominent role in the
  central nervous system, in particular in the
  brain. Nitric oxide is also important in the
  immune system, where it is used as a cell-killing
  agent.