Caenorhabditis elegans

•Free living nematode•1mm long and transparent•Lives in soil•Feed on microorganisms•E.coli in laboratory•Hermaphrodite sex•Rare males (0.05%)•Crossing•Eggs•Life span 2-3 weeks•Generation time 4 days•C.elegans field started in 1965 with Sydney Brenner•2002 Nobel prize•2003 C.elegans survived the Space Shuttle Columbia’s disintegration

first mitosis

gastrulation

Embryogenesis

first mitosis

gastrulation

Cell lineage

The developmental fate of every single somatic cell (959-1031) has mapped out

dauer

Life Cycle

C.elegans anatomy

spermatheca

cuticle

•Cheap•Life cycle is 4 days•Genome completely sequenced (100X6 bps)•A lot of information is available in web•19.000 genes: very little genes redundancy•Low complexity but with organs and tissue specifications•Transparent (anatomy)•Hermaphroditic lifestyle, males available for crossing

•Biochemistry difficult•No cell lines available•dissection of specific tissues is unrealistic

WHY C.elegans?

Methods

C.elegans and the Web

Information about C.elegans is stored in the database ACeDB.Several windows:1) Sequence window (genome as a string of nucleotide bases)2) Physical map window (genome as a set of DNA clones)3) Genetic window (genes as detected by mutation)

In addition in AceDB there are information about:1) Cell lineage and development2) ESTs, gene structure and homologies3) Genetic rearrangement and mutants available4) C.elegans meetings’ abstracts and publications.

deletion: 848 bp deletion CTCGATTT/ACCCCTGAACMutant phenotype: homozygous viable

CGC center

WT and mutant stocks of C.elegans are available fromthe Caenorhabditis Genetic Center (University of Missouri,Columbia)

Long term storage of C.elegans in liquid nitrogen (or -80C) is possible through the use of glycerol-containing media

Transformation

Transformation was introduced in the early 1980s. DNA is injected into the cytoplasm of the gonads. The DNA can pass through the germline in the form of extrachromosomal arrayPurposes:1) identification of genes by rescuing a mutant phenotype using a WT copy of the gene2) Expression pattern using the gene of interest with reporter3) Interference of a biological process by overexpression of WT or mutated gene

gonad40XDIC

Gene expression

3 approaches to study gene expression in C.elegans:

1) Reporter-gene fusion with transformation ( GFP, LacZ)2) In situ hybridization using mRNA3) Immunofluorescence with specific antibody

Genetics in C.elegans

Forward genetic1) R. Mutagenesis2) Transposons

Reverse Genetics1) RNAi2) PCR identification of rearrangements

Phenotypes genes Gene phenotypes

Forward Genetic

•Random mutagen (EMS/TMP-UV) to generate point mutations or small deletions•Analysis of F2 for the selected phenotypes•Mapping using visible markers and polymorphic DNA sequences

Gene targeting techniques based on Homologous Recombination are not available in C.elegans

Random mutagenesis

Forward Genetic

•Transposons: discrete segment of DNA moving in the genome, encoding a transposase•Normally present in C.elegans in different copies (strain-dependent) •Activated by forced expression of transposases •Most common:Tc1 (“cut and past mechanism”)•Insertional mutagenesis with Tc1 will generate mutant alleles tagged by the transposon that can be used for identify the mutated gene

Problems:1) Other Tc elements are mobilized in the mutator strain2) Several copies of the Tc1 (identification the mutagenic insertion)3) Transposition cannot be controlled

Solution: mobilization of Mos-1 element (a Tc1 absent in C.elegans) achieved by conditional expression of the Mos-1 transposase

Transposons

Reverse genetics

• RNA interference • Specific KO

KO/RNAi gene

Phenotype(s)

http://celeganskoconsortium.omrf.org/http://shigen.lab.nig.ac.jp/c.elegans/index.jsp

Specific KO:EMS/TMP mutagenesis (deletions)PCR to identified the mutated gene

RNA interference

Genome-scaleRNAi analysis

Double stranded RNA is used3 ways to interfere:1) Injection of dsRNA in gonads2) Soaking animals in dsRNA3) Feeding animal with bacteria producing dsRNA

http://www.geneservice.co.uk/products/rnai/Celegans.jspC.elegans RNAi library of about 16.000 genes

It is a “transient” KOWorks fine but not alwaysCan give interesting phenotypes when the KO is lethal

Is C.elegans a good model system to study endocytosis?

oocytes

Nerve system

Also: coelomocytes for Fluid fase endocytosis

Forward Genetic screenings to identify endocytic proteins

1) Yolk-GFP uptake in oocytes Identification of rme genes

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Identification of several rme genes (receptor-mediated endocytosis)Several genes were not identified in human (rme-1, rme-6, rme-8)Still several to be identified

Forward Genetic screenings to identify endocytic proteins

2) Compensatory endocytosis at presynaptic level: Identification of rics genes(resistant to inhibitor of cholinesterase)

Screening in presence of aldicarb allowed the identification of endocytic proteins such AP180, Synaptojanin, Endophilin, Synaptotagmin...

More complex screening because it targets proteins involved also in exocytosis (synaptotagmin, unc 13-18, syntaxin...) and production/transport of acetylcholine (kinesins)

cholinesterase

aldicarb

Reverse genetic screening to identify genes required for synapse structure and function

(Nature 2005)

• Pre-selected 2027 genes on the basis of sequence and domain and involvement in signal transduction, membrane trafficking synaptic localization

• Screening for aldicarb resistance by feeding RNAi, using eri-1 or eri-1;dgk-1 strains (aldicarb hypersensitive)

• 185 genes identified to be RIC (resistant to inhibitors of cholinesterase), 132 not known to be involved in synaptic transmission

• Expression pattern of 100 genes using transgenic animals (26 axonal proteins)

• presynaptic localization: Co-localization with synaptobrevin (24/26)

• Synaptic structure: distribution of GFP::SNB in the mutants• Molecular mechanisms????

EH1 EH2 EH3 COIL DPFs

EH1 EH2 EH3 COIL DPFs

Eps15

EHS-1

EHs COIL DPFs

TGAATGSL1ehs-1

Generating EPSF...Generating EPSF...Generating EPSF...Generating EPSF...Generating EPSF...

Generating EPSF...Generating EPSF...Generating EPSF...Generating EPSF...

EHS-1 is the C.e homologue of Eps15

EHS-1 is a neuronal protein and localizes in synaptic vesicle-rich regions

WT ehs-1

ATG

SL1

TGA

12077 13807

1 2 3 4 5 7 8

ehs-1(ok146)

Characterization of ehs-1 mutant

larval stage ehs-1

genotype

No Aldicarb 0.2mM

Aldicarb

0.5 mM

Aldicarb

1.0 mM

Aldicarb

L2 +/+ .98 (176) .52 (204) .16 (215) 0 (218)

-/- .97 (165) .70 (172) * .66 (165) * .06 (142) *

L4 +/+ .98 (505) .58 (564) .21 (600) .03 (169)

-/- .98 (512) .80 (494) * .43 (534) * .15 (177) *

Aldicarb: acetylcholinesterase inhibitor

ehs-1 is involved in synaptic transmission

ehs-1 animals show:•aldicarb resistance•Unc phenotype at high temperature(at 30°C they become paralyzed)•Depletion of synaptic vesicles at not permissive temperature

Electron-microscopy of WT, ehs-1 mutants and DN transgenic worms at 15°C (left) and 30°C (right).Arrows indicate presynaptic zones where vesicles are recycled.

TS Uncoordinate phenotype of ehs-1

mutant

Genetic and Physical interaction with dynamin

GS

T-E

ps15

GS

T-G

rb

GS

TC

OS

lys

Pre

-E

ps15

m

-Eps

15 p

PC

12

lys

-E

ps15

m

-E

ps15

p

Pre C

GS

T

GS

T-E

ps15

Dyn

inpu

t

WB-dyn

•ehs-1;dyn-1 double mutant is almost lethal•EHS-1 interacts with DYN-1•hEps15 interacts with hDynamin

The TS uncoordinate phenotype of ehs-1 KO worms is similar to thedynamin mutant phenotype

ehs-1 +

ehs-1 is a positive regulator of dynamin function

Lesson from C.elegans:1) EHS-1 (and EPS 15) is a neuronal protein2) Involved in synaptic transmission3) Partner of dynamin

Conclusions

Great model system for genetic analysis (rapid life cycle,small size,easy to grow in lab,self fertilization, crossing with males)Small genome(no redundancy) and simple anatomy (1000 cells, transparent)Constant cell number in the same position make the animal suitable for studying development

For RME and fluid fase endocytosis several mutants were identified by genetic screening, at least 20 are still without name and identity.......

BRIC Biotech Research & Innovation Centre University of Copenhagen

Simon RoseClaudia KragAnna Schultz