142

UNIT 4.2

C. elegans Biology

Presented by: Dr. Nate Szewczyk

NEMATODES AND INSECTS

143

UNIT 4.2 C. elegans Biology Dr. Nate Szewczyk Vocabulary:

• 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.

NEMATODES AND INSECTS

144

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:

• High levels of noise • High levels of vibration

• High levels of CO2 • Variable temperature

• Variable pressure • Hyper-gravity

• Micro-gravity

NEMATODES & INSECTS

145

Spaceflight induces biological changes:

• Altered growth and metabolism: • Altered mammalian physiology: ◦ 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

(Human embryonic lung, Mouse osteoblasts) Increased size (HeLa) Reduced growth rate (Hybridoma) Altered cytoskeletal morphology (Mouse osteoblasts)

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) Muscle atrophy (Human, Rat) Bone loss (Human, Rat) Altered immune response (Human, Rat) Fluid shift (Human)

NEMATODES & INSECTS

146

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

NEMATODES & INSECTS

147

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

NEMATODES & INSECTS

148

Essentially Invariant Cell Lineage

Lineage studies led to the discovery of apoptosis 2002 Nobel prize in Physiology or Medicine

Studies also led to the discovery of vulval defects, which result from

alteration in signaling from the RAS pathway.

CED-4 ~ APAF-1 CED-9 ~ Bcl-2 CED-3 ~ Caspase

NEMATODES & INSECTS

149

�Wiring� of the Nervous System has been Mapped

Allows for Detection of �Silent� Mutations in the Nervous System

NEMATODES & INSECTS

150

Alternative �dauer� state found in the life cycle

Studies of the dauer Mutants led to the Discovery of TGF-β and Insulin-like Signaling in the Worm

Glucose based metabolism gene

NEMATODES & INSECTS

151

Multiple Receptors Control Protein Degradation in Muscles by Multiple Mechanisms

This is the only system for which we know this.

NEMATODES & INSECTS

152

History of Worms in Space Unique challenges of spaceflight experiments:

• Limited ability to oversee experiment • Use of non-standard laboratory equipment • Limited power • Limited size • Limited weight • Limited crew intervention

What are we doing?

• 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?

• 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

NEMATODES & INSECTS

153

Biocapatibility Material: Manufacturer:

Tubes

1 ml Eppendorf 15 ml conical Falcon 50 ml conical Falcon

Dishes 60 mm petri Falcon 100 mm petri Falcon 60 mm tissue culture Falcon 100 mm tissue culture Falcon 24 well tissue culture Corning 96 well tissue culture Corning

Flasks 25 cm2 cell culture Corning 75 cm2 cell culture Corning

Membranous devices 10 mm membrane tubing Spectrapor Opticell� BioCrystal FEP bag 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

NEMATODES & INSECTS

154

Effects on Metabolism

Lipid Stores

Protein Stores & General Appearance

NEMATODES & INSECTS

155

Gene Expression

Microarray

a) Downregulated in CeMM versus on NGM Decrease

(Fold log2)

Genepairs Name

Encodes or predicted to encode 2.12 ± 0.16 F59A1.10 Diacylglycerol acyltransferase 2 2.09 ± 0.17 C18C4.5 Myosin/CLIP-190 like 2.06 ± 0.07 T21B10.3 Nuclear protein 2.23 ± 0.16 F42H10.7 DGCR14/ES2 like 2.31 ± 0.22 K05C5.5 UTP4 like 2.23 ± 0.06 ZK856.9 Zinc finger 2.58 ± 0.15 ZC395.9 C2H2 Zinc finger 2.82 ± 0.23 T10F2.3 Ulp1 protease family 2.96 ± 0.29 ZK593.5 dnc-1 2.36 ± 0.15 F22B7.5 dnj-10 2.07 ± 0.12 Y46H3C.D DNA gyrase/topoisomerase 3.21 ± 0.32 T07A9.6 daf-18 2.70 ± 0.15 Y39A1A.12 AAA ATPase 2.27 ± 0.11 F49E8.2 Glutaredoxin 2.39 ± 0.16 ZC317.7 Dynactin interacting protein like 2.78 ± 0.17 T02B5.1 Carboxylesterase 2.44 ± 0.22 ZK1320.9 Acetyl-CoA hydrolase/transferase 3.90 ± 0.28 F25D1.1 Protein Phosphatase 2 C 3.88 ± 0.35 F56A6.1 PAZ, Piwi , & ncoil domains 2.46 ± 0.18 C55B7.1 glh-2 2.41 ± 0.20 C10H11.1 ncoil and villin headpiece domains 2.74 ± 0.15 K07D8.1 mup-4 4.90 ± 0.20 T22B7.7 Long-chain acyl-CoA thioester hydrolase 3.82 ± 0.38 F59D8.C vit-3 3.01 ± 0.24 C47B2.6 NAD dependent epimerase/dehydratase 3.63 ± 0.32 F02A9.5 Carboxyl transferase

NEMATODES & INSECTS

156

Gene Expression

Microarray

b) Upregulated in CeMM versus on NGM Increase

(Fold log2)

Genepairs Name

Encodes or predicted to encode 2.11 ± 0.11 R13H8.1a daf-16 2.33 ± 0.19 T20D3.6 Transmembrane protein 2.27 ± 0.16 F31E3.6 Transmembrane protein 3.95 ± 0.29 F35A5.2 Transmembrane protein 3.58 ± 0.10 C18A11.2 Transmembrane protein 2.46 ± 0.17 C39D10.2 Cytochrome c binding 2.31 ± 0.19 M03E7.4 LD-lipoprotein receptor 3.92 ± 0.36 T08G5.10 mtl-2 3.80 ± 0.21 7T10B10.6 Heavy metal binding 3.15 ± 0.09 F32H5.3 Transmembrane protein 2.15 ± 0.14 C25E10.8 Trypsin inhibitor like 2.85 ± 0.27 K11G9.6 mtl-1 2.14 ± 0.18 F18E9.2 nlp-7 7.64 ± 0.18 F36D3.9 Cysteine endo-peptidase 2.37 ± 0.20 F54E7.2 rps-12 2.28 ± 0.16 F37C12.9 rps-14 2.60 ± 0.20 F37C12.11 rps-21 2.10 ± 0.15 F39B2.6 rps-26 2.47 ± 0.16 B0414.4 rps-29 2.49 ± 0.12 Y48B6A.2 rpl-43 2.53 ± 0.13 F28F8.3 SNRNP-E like 2.84 ± 0.11 Y46G5.Z Unknown

NEMATODES & INSECTS

157

History of Worms in Space

STS-95 (E. Moss), Suspended in liquid: Animals died (of hypoxia?)

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-107 (C. Conley), Grown on NGM and CeMM: CeMM supported animals in flight Animals survived Columbia breakup

STS-65 (S. Nagaoka), Grown on ground: Free flow electrophoresis of DNA successful

NEMATODES & INSECTS

158

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 10 N2

NEMATODES & INSECTS

159

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

NEMATODES & INSECTS

160

Notes: