Transcript Document

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
Embryogenesis
first mitosis
gastrulation
Cell lineage
The developmental fate of every single somatic cell (959-1031)
has mapped out
first mitosis
gastrulation
Life Cycle
dauer
C.elegans anatomy
cuticle
spermatheca
WHY C.elegans?
•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
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 development
2) ESTs, gene structure and homologies
3) Genetic rearrangement and mutants available
4) C.elegans meetings’ abstracts and publications.
deletion: 848 bp deletion CTCGATTT/ACCCCTGAAC
Mutant phenotype: homozygous viable
CGC center
WT and mutant stocks of C.elegans are available from
the Caenorhabditis Genetic Center (University of Missouri,
Columbia)
Long term storage of C.elegans in liquid nitrogen (or -80C)
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 array
Purposes:
1) identification of genes by rescuing a mutant phenotype using
a WT copy of the gene
2) Expression pattern using the gene of interest with reporter
3) Interference of a biological process by overexpression of
WT or mutated gene
gonad
40X
DIC
Gene expression
3 approaches to study gene expression in C.elegans:
1) Reporter-gene fusion with transformation ( GFP, LacZ)
2) In situ hybridization using mRNA
3) Immunofluorescence with specific antibody
Genetics in C.elegans
Forward genetic
1) R. Mutagenesis
2) Transposons
Phenotypes
genes
Reverse Genetics
1) RNAi
2) PCR identification
of rearrangements
Gene
phenotypes
Forward Genetic
Gene targeting techniques based on Homologous
Recombination are not available in C.elegans
Random mutagenesis
•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
Forward Genetic
Transposons
•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 strain
2) 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
Reverse genetics
KO/RNAi gene
Phenotype(s)
•
•
RNA interference
Specific KO
Specific KO:
EMS/TMP mutagenesis (deletions)
PCR to identified the mutated gene
http://celeganskoconsortium.omrf.org/
http://shigen.lab.nig.ac.jp/c.elegans/index.jsp
RNA interference
Double stranded RNA is used
Genome-scale
3 ways to interfere:
RNAi analysis
1) Injection of dsRNA in gonads
2) Soaking animals in dsRNA
3) Feeding animal with bacteria producing dsRNA
http://www.geneservice.co.uk/products/rnai/Celegans.jsp
C.elegans RNAi library of about 16.000 genes
It is a “transient” KO
Works fine but not always
Can 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
Qui ckTi me™ and a
TIFF (LZW) decompressor
are needed to see thi s picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (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)
aldicarb
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)
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????
EHS-1 is the C.e homologue of Eps15
Eps15
EH1 EH2 EH3
EHS-1
EH1 EH2
EH3
COIL
COIL
EHs
ehs-1
DPFs
DPFs
COIL
DPFs
SL1 AT G
T GA
EHS-1 is a neuronal protein and localizes
in synaptic vesicle-rich regions
Gener at ing EPSF. . .
Gener at ing EPSF. . .
Characterization of ehs-1 mutant
ehs-1(ok146)
12077
13807
SL1
ATG
1
2
3
4
5
7
8
T GA
WT
ehs-1
Aldicarb: acetylcholinesterase inhibitor
larval stage
ehs-1
No Aldicarb
genotype
L2
L4
0.2mM
0.5 mM
1.0 mM
Aldicarb
Aldicarb
Aldicarb
+/+
.98 (176)
.52 (204)
.16 (215)
0 (218)
-/-
.97 (165)
.70 (172) *
.66 (165) *
.06 (142) *
+/+
.98 (505)
.58 (564)
.21 (600)
.03 (169)
-/-
.98 (512)
.80 (494) *
.43 (534) *
.15 (177) *
ehs-1 is involved in synaptic transmission
TS Uncoordinate
phenotype of ehs-1
mutant
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.
Genetic and Physical interaction with dynamin
The TS uncoordinate phenotype of
ehs-1 KO worms is similar to the
dynamin mutant phenotype
•ehs-1;dyn-1 double mutant is almost lethal
•EHS-1 interacts with DYN-1
•hEps15 interacts with hDynamin
Dyn input
PC12 lys
GST-Grb
COS lys
WB a-dyn
ehs-1 is a positive regulator of dynamin function
ehs-1
+
Lesson from C.elegans:
1) EHS-1 (and EPS 15) is a neuronal protein
2) Involved in synaptic transmission
3) 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 Rose
Claudia Krag
Anna Schultz