1. 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

2. first mitosis gastrulation Embryogenesis

3. first mitosis gastrulation Cell lineage The developmental fate of every single somatic cell (959-1031) has mapped out

4. dauer Life Cycle

5. C.elegans anatomy spermatheca cuticle

6. Cheap Life cycle is 4 days Genome completely sequenced (100 X6 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?

7. 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.

8. deletion: 848 bp deletionCTCGATTT/ACCCCTGAAC Mutant phenotype: homozygous viable

9. 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

10. 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

11. 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

12. Genetics in C. elegans Forward genetic 1) R. Mutagenesis 2) Transposons Reverse Genetics 1) RNAi 2) PCR identification of rearrangements Phenotypes genes Gene phenotypes

13. 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

14. 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 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 Transposons

15. 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

16. RNA interference Genome-scale RNAi analysis Double stranded RNA is used 3 ways to interfere: 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

17. Is C.elegans a good model system to study endocytosis? oocytes Nerve system Also: coelomocytes for Fluid fase endocytosis

18. Forward Genetic screenings to identify endocytic proteins 1)Yolk-GFP uptake in oocytes Identification of rme genes 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

19. 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

20. 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????

21. Eps15 EHS-1 ehs-1 EHS-1 is the C.e homologue of Eps15 EHS-1 is a neuronal protein and localizes in synaptic vesicle-rich regions

22. WT ehs-1 ehs-1(ok146) Characterization of ehs-1 mutant Aldicarb: acetylcholinesterase inhibitor ehs-1 is involved in synaptic transmission

23. 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

24. Genetic and Physical interaction with dynamin GST-Eps15 GST-Grb GST COS lys Pre  -Eps15 m  -Eps15 p PC12 lys  -Eps15 m  -Eps15 p Pre C GST GST-Eps15 Dyn input 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 the dynamin mutant phenotype

25. ehs-1 + ehs-1 is a positive regulator of dynamin function Lesson from C.elegans: 1)EHS-1 (and EPS 15) is a neuronal protein 2)Involved in synaptic transmission 3)Partner of dynamin

26. 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.......

27. BRIC Biotech Research & Innovation Centre University of Copenhagen Simon Rose Claudia Krag Anna Schultz