UNIT 4.2
C. elegans Biology
Presented by:
Dr. Nate Szewczyk
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NEMATODES AND INSECTS
UNIT 4.2
C. elegans Biology
Dr. Nate Szewczyk
Vocabulary:
•
•
•
•
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Apoptosis
Autosomal
Convergent
Gene
Prokaryotic
Purpose:
The NASA Fundamental Space Biology Program has selected several model organisms to be
used for future spaceflight research efforts. C. elegans is among the first models selected for
near-term spaceflight opportunities. This workshop will provide an introduction to basic C.
elegans biology and an overview of results from past flights.
Objectives:
a)
Describe basic C. elegans culture conditions.
b) Describe the basic life-cycle of C. elegans.
c)
Explain why C. elegans is a model system.
d) Recall results from past C. elegans flights.
e)
List sources of information on C. elegans.
f)
Imagine C. elegans experiments consistent with NASA’s Research Goals.
g) Invent a hardware design for a C. elegans spaceflight experiment.
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NEMATODES AND INSECTS
Why?
A NASA Vision
To improve life here, to extend life to there, to find life beyond
To prepare for and hasten the journey, the following questions must be answered through
research:
• How can we assure survival of humans traveling from earth?
• What must we know about how space changes life forms, so that humankind with
flourish?
• What new opportunities can our research bring to expand understanding of the laws of
nature and enrich lives on Earth?
• What technology must we create to enable the next explorers to go beyond where we
have been?
• How can we educate and inspire the next generations to take the journey?
Spaceflight involves numerous stressors:
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High levels of noise
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High levels of vibration
•
High levels of CO2
•
Variable temperature
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Variable pressure
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Hyper-gravity
•
Micro-gravity
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NEMATODES & INSECTS
Spaceflight induces biological changes:
• Altered growth and metabolism:
◦ Prokaryotic:
Increase in phage production (E. coli)
Increased resistance to high dose radiation
(Salmonella, E. coli)
Increased growth rate (P. vulgaris)
Increased antibiotic resistance (E. coli)
◦ Mammalian cells:
Decreased glucose utilization
• Altered mammalian physiology:
Reduced mRNA synthesis (Rat)
Reduced growth rate in blood cells
(Rat)
Altered hormone levels (Human, Rat)
Altered tubulin/cytoskeletal synthesis
(Rat)
Altered collagen distribution/secretion
(Rat)
(Human embryonic lung, Mouse osteoblasts)
Increased size (HeLa)
Reduced growth rate (Hybridoma)
Altered cytoskeletal morphology
(Mouse osteoblasts)
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Muscle atrophy (Human, Rat)
Bone loss (Human, Rat)
Altered immune response (Human, Rat)
Fluid shift (Human)
NEMATODES & INSECTS
Why worms?
Caenorhabditis elegans “the worm” is a model system for biology.
Advantages:
• Established research community
o Over 150 individual labs study C. elegans
o Online resources
• Small
o Adults are roughly 1 mm long
• Easy to grow and manipulate large numbers of worms
o Need an incubator and agar plates with bacterial lawns
o Self fertilize, thus easy to maintain genetic homozygotes
• Rapid life cycle
o Egg to adult is roughly 2 days at 25°C
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NEMATODES & INSECTS
Caenorhabditis elegans “the worm” is a model system for biology.
Advantages:
• Genetically tractable
o Males can be used to mate in or out genes of
interest
o Has a well-defined genetic map
− Mutants readily available - CGC
− Five pairs of autosomal and one pair of
sex chromosomes
o Has a sequenced genome
− Available on-line
− Allows “reverse” genetics
• Well studied
o Most techniques can be done:
PCR
Northerns
IIF
Southerns
Transgenics
RNA-i
Westerns
Drugs/Inhibitors
Essentially Invariant Cell Lineage
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NEMATODES & INSECTS
Essentially Invariant Cell Lineage
Lineage studies led to the discovery of apoptosis
2002 Nobel prize in Physiology or Medicine
CED-4 ~ APAF-1
CED-9 ~ Bcl-2
CED-3 ~ Caspase
Studies also led to the discovery of vulval defects, which result from
alteration in signaling from the RAS pathway.
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NEMATODES & INSECTS
“Wiring” of the Nervous System has been Mapped
Allows for Detection of “Silent” Mutations in the Nervous System
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NEMATODES & INSECTS
Alternative “dauer” state found in the life cycle
Studies of the dauer Mutants led to the Discovery of TGF-β and Insulinlike Signaling in the Worm
Glucose based metabolism gene
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NEMATODES & INSECTS
Multiple Receptors Control Protein Degradation
in Muscles by Multiple Mechanisms
This is the only system for which we know this.
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NEMATODES & INSECTS
History of Worms in Space
Unique challenges of spaceflight experiments:
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Limited ability to oversee experiment
Use of non-standard laboratory equipment
Limited power
Limited size
Limited weight
Limited crew intervention
What are we doing?
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Developed a new culture system for worms in flight
Have been using the system to look at the effects of hyper gravity on earth
Attempting to use the system in flight to:
o Establish baseline behavioral data for C. elegans in-flight
o Establish baseline gene expression data for C. elegans in-flight
o Examine the use of C. elegans as a model for space flight atrophy
Working with SSBRP to establish C. elegans as a model system in space
Why the Culture System?
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•
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Traditional method of growing worms subjects them to surface tension forces of
10,000-100,000xG. (masks microG?)
Traditional method requires frequent astronaut intervention.
New culture system avoids issue of surface tension and allows automation.
o Animals grow for at least four weeks in unchanged media.
Technical Issues with Worms Grown in Cemm:
• Tested ability to perform standard lab protocols
○ Dauer formation/recovery
○ Freezing
○ Manipulation of animals
○ Synchronizing populations (bleaching, gravity)
○ Mating
• Tested differences in form of medium (liquid vs. solid)
• Examined ability of animals to ‘condition’ the medium
○ pH change
○ Accumulation of shed cuticle and dead animals
• Biocompatibility of various equipment with animals in medium
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NEMATODES & INSECTS
Biocapatibility
Material:
Manufacturer:
Tubes
1 ml
15 ml conical
50 ml conical
Eppendorf
Falcon
Falcon
Dishes
60 mm petri
100 mm petri
60 mm tissue culture
100 mm tissue culture
24 well tissue culture
96 well tissue culture
Falcon
Falcon
Falcon
Falcon
Corning
Corning
Flasks
25 cm2 cell culture
75 cm2 cell culture
Corning
Corning
Membranous devices
10 mm membrane tubing
Opticell™
FEP bag
Spectrapor
BioCrystal
American Fluoroseal
How do worms grown in CeMM compare to those on NGM?
Developmental profile
Growth Rate
Larval Stages
Effects on metabolism
General appearance
Lipid stores
Protein stores
Reproductive profile
Brood Size
Egg Laying period
Gene expression
Microarray
Life span
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NEMATODES & INSECTS
Effects on Metabolism
Lipid Stores
Protein Stores & General Appearance
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NEMATODES & INSECTS
Gene Expression
Microarray
a) Downregulated in CeMM versus on NGM
Decrease
(Fold log2)
2.12 ± 0.16
2.09 ± 0.17
2.06 ± 0.07
2.23 ± 0.16
2.31 ± 0.22
2.23 ± 0.06
2.58 ± 0.15
2.82 ± 0.23
2.96 ± 0.29
2.36 ± 0.15
2.07 ± 0.12
3.21 ± 0.32
2.70 ± 0.15
2.27 ± 0.11
2.39 ± 0.16
2.78 ± 0.17
2.44 ± 0.22
3.90 ± 0.28
3.88 ± 0.35
2.46 ± 0.18
2.41 ± 0.20
2.74 ± 0.15
4.90 ± 0.20
3.82 ± 0.38
3.01 ± 0.24
3.63 ± 0.32
Genepairs Name
F59A1.10
C18C4.5
T21B10.3
F42H10.7
K05C5.5
ZK856.9
ZC395.9
T10F2.3
ZK593.5
F22B7.5
Y46H3C.D
T07A9.6
Y39A1A.12
F49E8.2
ZC317.7
T02B5.1
ZK1320.9
F25D1.1
F56A6.1
C55B7.1
C10H11.1
K07D8.1
T22B7.7
F59D8.C
C47B2.6
F02A9.5
Encodes or predicted to encode
Diacylglycerol acyltransferase 2
Myosin/CLIP-190 like
Nuclear protein
DGCR14/ES2 like
UTP4 like
Zinc finger
C2H2 Zinc finger
Ulp1 protease family
dnc-1
dnj-10
DNA gyrase/topoisomerase
daf-18
AAA ATPase
Glutaredoxin
Dynactin interacting protein like
Carboxylesterase
Acetyl-CoA hydrolase/transferase
Protein Phosphatase 2 C
PAZ, Piwi , & ncoil domains
glh-2
ncoil and villin headpiece domains
mup-4
Long-chain acyl-CoA thioester hydrolase
vit-3
NAD dependent epimerase/dehydratase
Carboxyl transferase
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NEMATODES & INSECTS
Gene Expression
Microarray
b) Upregulated in CeMM versus on NGM
Increase
(Fold log2)
2.11 ± 0.11
2.33 ± 0.19
2.27 ± 0.16
3.95 ± 0.29
3.58 ± 0.10
2.46 ± 0.17
2.31 ± 0.19
3.92 ± 0.36
3.80 ± 0.21
3.15 ± 0.09
2.15 ± 0.14
2.85 ± 0.27
2.14 ± 0.18
7.64 ± 0.18
2.37 ± 0.20
2.28 ± 0.16
2.60 ± 0.20
2.10 ± 0.15
2.47 ± 0.16
2.49 ± 0.12
2.53 ± 0.13
2.84 ± 0.11
Genepairs Name
R13H8.1a
T20D3.6
F31E3.6
F35A5.2
C18A11.2
C39D10.2
M03E7.4
T08G5.10
7T10B10.6
F32H5.3
C25E10.8
K11G9.6
F18E9.2
F36D3.9
F54E7.2
F37C12.9
F37C12.11
F39B2.6
B0414.4
Y48B6A.2
F28F8.3
Y46G5.Z
Encodes or predicted to encode
daf-16
Transmembrane protein
Transmembrane protein
Transmembrane protein
Transmembrane protein
Cytochrome c binding
LD-lipoprotein receptor
mtl-2
Heavy metal binding
Transmembrane protein
Trypsin inhibitor like
mtl-1
nlp-7
Cysteine endo-peptidase
rps-12
rps-14
rps-21
rps-26
rps-29
rpl-43
SNRNP-E like
Unknown
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NEMATODES & INSECTS
History of Worms in Space
STS-42 (G. Nelson), Grown on NGM:
Males mate
Undergo two generations in-flight
No gross morphologic defects
Increased rate of mutation
STS-76 (G. Nelson), Suspended in M9:
Radiation, not microgravity, key factor
in increased rate of mutation
STS-65 (S. Nagaoka), Grown on ground:
Free flow electrophoresis of DNA
successful
STS-95 (E. Moss), Suspended in liquid:
Animals died (of hypoxia?)
STS-107 (C. Conley), Grown on NGM and CeMM:
CeMM supported animals in flight
Animals survived Columbia breakup
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NEMATODES & INSECTS
Actual Design
HOBO:
Autonomous temperature
recorder, temperature logged
every 30 minutes
LiOH:
Used to ‘soak up’ CO2
N2:
Weight on NGM
CC1
Weight on CeMM
ms:
Mixed Stage
Can 1
Can 2
Can 3
Can 4
Can 5
Can 6
HOBO
HOBO
500ms CC1
LiOH
HOBO
LiOH
500 CC1
LiOH
500ms CC1
500ms CC1
10 N2
10 N2
10 N2
500ms CC1
500ms CC1
500ms CC1
10 N2
10 N2
100 CC1
10 N2
500ms CC1
500ms CC1
10 N2
10 N2
100 N2
100 CC1
500ms CC1
500ms CC1
100 N2
10 N2
10 CC1
100 N2
500ms CC1
500ms CC1
10 N2
10 N2
500 N2
10 CC1
500ms CC1
500ms CC1
500N2
10 N2
500ms CC1
500 N2
500ms CC1
500ms CC1
10 N2
10 N2
500ms CC1
500ms CC1
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10 N2
NEMATODES & INSECTS
Reference Materials
Papers on Worms in Space:
Johnson, T.E., and Nelson, G.A. Caenorhabditis elegans: A model system for space
biology studies. Experimental Gerontology 26:299-309 1991
Nelson, G.A., et al. Nematode radiobiology and development in space. Results from
IML-1.
Proceedings of the Fifth European Symposium on Life Sciences Research in Space.
187-91 1994
Nelson, G.A., et al. Radiation effects in nematodes: Results from IML-1 experiments.
Adv. Space Res. 14: 87-91 1994
Nelson, G.A., Schubert, W.W., Kazarians, G.A., and Richards, G.F. Development and
chromosome mechanics in nematodes: Results from IML-1. Adv. Space Res. 14: 209-14
1994
Hartman, P.S., et al. A comparison of mutations induced by accelerated iron particles
versus
those induced by low earth orbit space radiation in the FEM-3 gene of Caenorhabditis
elegans. Mutation Research 474: 47-55 2001
Books on Worms:
The Nematode Caenorhabditis elegans
Wood, W.B., Ed. Cold Spring Harbor Laboratory Press 1988
C. elegans II
Riddle, D.L., Blumenthal, T., Meyer, B., and Priess, J.R., Eds.
Cold Spring Harbor Laboratory Press 1997
Caenorhabditis elegans: modern biological analysis of organism
(Methods in Cell Biology , Vol 48)
Epstein, H.F. and Shakes, D.C., Eds. Academic Press 1995
C. elegans (A practical approach)
Hope, I.A., Ed. Oxford University Press 1999
Online Resources:
http://elegans.swmed.edu
http://www.wormbase.org
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NEMATODES & INSECTS
Notes:
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