Presentation on theme: "C. elegans as a Model System"— Presentation transcript:
1C. elegans as a Model System C. elegans: introduction (anatomy, life cycle, genome and etc.)Essentially invariant lineage of C. elegans.Cell death as a cell fate.Genes that affect lineage specification and timing: link to miRNAs.RNAi discovery.miRNAs and RNAi.
2C. elegans timeline. Developed by Sydney Brenner (1963) Mutants published by Brenner (1974)1976 postembryonic cell lineages determined (Sulston and Horwitz)1982 "Programmed cell death" (Horwitz et al.) (Nobel Prize Brenner, Sulston, Horwitz 2002)1983 complete embryonic cell lineages determined (Deppe et al., Sulston et al.)1986 Complete connectivity of nervous system established (White et al.) "The mind of a worm”RNAi and miRNA discovered in worms. (Nobel Prize: Fire, Mello 2006)First use of GFP in animals (Novel Prize: Chalfie, 2008)1998 First animal genome sequenced (97Mb, now 100.3Mb)This is about 1/30 the size of the human genome (3 Gb). C. elegans have about 20,000 coding sequence genes, more than half that of humans (30,000-40,000 genes). First animal to be sequenced! Knowing the sequence allows genes of interest to be easily cloned. Also it opened opportunity for reverse genetic approach.
3C. (Caenorhabditus) elegans 1st introduced and used by Sydney Brenner (1963)to study development and neurology.Adults are ~1mm long (small).They can be grown on agarplates with lawn of bacteria.They have a short generationtime- 3 days from egg-layingto adulthood, brood size > 300.They are transparent, so internalanatomy can be easily observed.Goldstein lab
4Caenorhabditis elegans 959 somatic cellsXX karyotype5 autosomes I-V and X (6 total)XX animals become hermaphroditesXO animals become males(chromosome non-disjunction)With one less chromosome, males develop79 more neurons and 25 more muscle cellsthan hermaphrodites, used for mating.Hermaphrodite produces many progeny---300 worms!Adult hermaphrodites have 959 somaticcells while males have 1031.XO karyotype1031 cells
5C. elegans morphology Organs and tissues Epidermal system Digestive systemReproductive systemMuscular systemExcretory systemNervous systemMuscles
6Nervous system By comparison 105 neurons in flys, ~300 in worms Hermaphrodite 302 neurons (118 types)5000 chemical synapses2000 neuromuscular junctionsMale neuronsBy comparison105 neurons in flys, ~300 in wormsInterneurons and motorneuronsMajor neurotransmitters:Ach -exitatoryGABA- inhibitorySerotonin (HSN neuron- egg laying)DopamineFMRF amide peptidesWood W.B The Nematode Caenorhabditis elegans
7Life cycle of Caenorhabditis elegans Proliferationgeneration of embryonic founder cellsbulk of cell divisions and gastrulation.a spheroid of cells:ectoderm - hypodermis and neurons,mesoderm - pharynx and muscle,endoderm - germline and intestine.Organogenesisterminal differentiation3 days at 22C
8C. elegans cell lineageOne amazing advantage of worms is that every worm has the exact same number of somatic cells!Hatching larva=558 cellsMales (XO)=1031 somatic cells+ ~1000 spermHermaphrodite (XX)= 959 somatic cells+~2000 eggs and sperm.Somatic cells arise by an INVARIANT cell lineage.The divisions are invariant in pattern, timing, andorientation of each division.
9Here it is: C. elegans cell lineage EMSP1P2P3Know how to read a lineage diagram!1) Each branch indicates a cell division2) When a cell differentiates and no longer divides, line ends3) Line ending in X= programmed cell death4) Moving from top to bottom is moving ahead in time
10Postembryonic cell lineage was completed first. In the presence of food, cell divisions resume and postembryonic developmental program begins 3 hours after hatching. a first stage larva has approximately 671 cells, 113 undergo programmed death in the course of development. About 10% of the remaining 558 cells in a newly hatched larva (51 in hermaphrodites, 55 in the male), are blast cells that divide further.DivisionsMigrationsDifferentiationDeath
11Direct observations are possible Cell divisions.Sequential photographs of an L1 hermaphrodite, lateral view; Nomarski optics vcn, ventral cord neurons.0 min, interphase;16 and 21 min, P10 prophase;24 min, P10 metaphase;26 min 10 anaphase;27 mm, P10 telophase;29 min, P9 prophase;33 and 34 min, P9 metaphase.Sulston & Horvitz. Developmental Biology 56: (1977)
12Founder cell derivation by asymmetric cell division and inductions Founder cells: AB, MS, E, C, D and P4.Distinctive properties of founder cells defined by:division ratenature of their progeny
13Germline formation in C. elegans 6 Founder Cells+98 die+14 dieSmall posterior cell goesgermline+1 dies
14How are invariant lineages established and maintained? Partitioning of prexisting maternal componentsCell-autonomous programsCell-cell interactions
15Asymmetric cell division Sperm entry defines posterior pole. Sperm aster microtubules organize microfilaments needed to establish asymmetries.Several genes identified that are required for this partitioning of material and are called par genes in worms. Differential partitioning of maternally expressed proteins to anterior and posterior of egg occur: PAR-1, -2 associate with posterior surface; PAR-3, -6, PKC-3 associate with anterior surface. They coordinate the polarization of cytoskeleton affecting distribution of other cell components.P granules(ribonucleoprotein particals) segregate to posterior pole until 16-cell stage and P4, just one cell, will give a rise to germline.A-P partitioning occurs in all germline precursors.
16Early inductions P2-EMS 1st transcription occurs at 4-cell stage in EMS.Can block transcription get to 100 cell stageLineage commitment can occur through multiple mechanismsP2 induces ABp but not ABa by contactP2 induces EMS asymm. division.
17Where do specific tissues come from? Lineages do not strictly produce single tissue types.ABMSECDP4While intestine & germ-line come from single founder cells, the muscle, nervous system & skin cells each arise from multiple lineages. This means, for example, that muscles do not have one “founder” cell early in development.
18Cell death can be a genetically programmed cell fate. Indicate death eventThe Nematode Caenorhabditis Elegans.1988. Editor William B. Wood ,Cold Spring Harbor Laboratory.Specific cells with diverse developmental origins undergo programmed cell death at specifictimes during development.Programmed cell death is characterized by a series of specific morphological changes.There must be genes that control both the decision to express that fate and the execution.
19Genetics of Programmed Cell Death: CED-3, CED-4 and CED-9 have human counterparts.A ced-1 has defect in the engulfment of dyingcells. Programmed cell death does not occur ina ced-3 mutant. ced-3 (caspase) is a killer.egl-1 (dead HSN neuron)Search for supressionof egl (HSN restored)ced-4 (Apaf-1-like-protein) is a killer.The sister of NSM neuron in the pharynxsurvives instead of dyingced-9 (gf)ced-9 has similarities to theproto-oncogene Bcl-2(B cell lymphoma)
20The genetic pathway for programmed cell death in C. elegans
21The principle of biological universality “One point that emerges from the studies of programmedcell death in C. elegans and other organisms is the strikingsimilarity of genes and gene pathways among organisms thatare as superficially distinct as worms and humans...I like to refer to this theme as “The principle of biologicaluniversality,”and it underlies my strong conviction that the rigorous,detailed and analytic study of the biology of any organism islikely to lead to findings of importance in the understandingof other organisms.”
22miRNAs miRNAs were discovered in C. elegans. Additionally, much of what we know about miRNAsand their targets has come from studies in C. elegans.C. elegans has over 200 miRNAs. Humans have over 1000.Most biological processes are touched in some way by miRNAs.The excitement generated by the discovery ofmiRNAs, and their similarity to siRNAs, has made ahuge impact on our thinking about genetic control.
23Genes that affect timing and Discovery of microRNAs (miRNAs) The definition of heterochrony is a changein the relative timing of developmental events. Eachscale of development, the cycle of cell divisions, the growthof tissues, the formation of organs, requires proper timing.Two general phenotypes are seen in heterochronicmutants —‘precocious,’ in which developmental events are skipped,‘retarded,’ in which they are repeated.A heterochronic mutation may affect different tissues:intestine, epidermis, muscle, and neurons, anddifferent kinds of developmental events:a pattern of cell division, a cell cycle lengths, anddifferentiation.
24An example: lin-4 and lin-14 mutants Hypodermal cell lineageMoss E Current Biology, R425.Lin-4 regulates transition from L1 to L2 stage.Lin-14 gene is a regulator of transcription.
25Lin-4 is the first microRNA gene. lin-4 encodes a small RNA molecule, a rare 61 nt pre-lin-4 precursor that is processed into a 22nt miRNA. These hairpin precursor is a characteristic feature of the miRNA class of regulatory RNAs.imperfectly base-paired to complementarysequences on target messengerOne of lin-4’s target genes, lin-14, encodes a novel nuclear protein and is a putative transcription factor. The lin-4 microRNA regulates lin-14 through specific sequences in the 3’UTR of the lin-14 mRNA.Upon lin-4 expression, lin-14 protein levels are reduced. Although transcription from the lin-14 gene still occurs, it is of no consequence.(Posttranscriptional control).Ambros V Nature 431,
26The conservation of let-7 across animal phylogeny showed that microRNAs are not exclusive to C. elegans.let-7 controls the L4-to-adult transition in C. elegans. lin-41 is one of the targets. let-7 mutants fail to execute the L4-to-adult transition (the seam cells fail to terminally differentiate and continue to divide), and die by bursting through the vulva (Reinhart et al., 2000).Moss E Current Biology, R425.
27Temporal regulation of the miRNAs lin-4 and let-7 and their target genes.Vella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook ,MicroRNAs increase in abundance at each stage and repress specific targets that encode developmental regulators. The change in regulators at each stage leads to a succession of developmental events.
28MicroRNA functions in animals and disease. E. Wienholds, R.H.A. Plasterk / FEBS Letters 579 (2005) 5911–5922
29miRNA-mediated repression of translation Nelson T TRENDS in Genetics, 23: 243.Target mRNAs are recognized by miRNAs in the form of ribonucleoprotein complexes (miRNPs), through sequence complementarity. This interaction can have direct and indirect effects on translation:1) Direct effects through inhibition of initiation of translation, which results in prevention of ribosome association with the target mRNA, or through inhibition of translation post-initiation. In the case of post-initiation repression – which includes premature ribosome drop off, slowed or stalled elongation, and cotranslational protein degradation – the repressed mRNA seems to be present in polysomes.2) miRNPs can have other effects on targeted mRNAs, including promoting deadenylation, which might result in degradation (increased turnover). It might take place in P bodies (denoted by P), which are enriched for factors involved in mRNA degradation.3) targeted mRNAs could be sequestered from the translational machinery and degraded or stored for subsequent use.
30RNAi (RNA interference) RNAi is an evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homologous mRNA sequences.1991 Fire et al. showed that antisense RNA could inhibit gene expression when injected into worms.Didn't always work!!!!!several papers showed that either antisense or sense RNA could interfere (Guo and Kemphues Cell:81,1995)1998 Fire et al. and the Mello lab showed that combination of sense and antisense worked all the time on certain genes. Probably got hairpins with sense or antisense alone.1998 Effect is post-transcriptional, enhanced degradation of target transcript.1999 First genes identified for RNAiEssential for transposon silencing.2000 Dicer enzyme cleaves dsRNA precursors to 21-23bp duplex oligonuc.
31systemic amplified heritable Important characteristics of RNAi in C. eleganssystemic amplified heritableWays to introduce dsRNA into worms:InjectionSoakingfeeding
32Plasmids for feeding Large-scale screens for RNAi deficient mutants. Timmins L. & Fire A Nature, 395:854.Large-scale screens for RNAi deficient mutants.Production of genome wide collections of RNAi feeding constructsSegments were cloned between flanking copies ofbacteriophage T7 promoter into a bacterial plasmid vector.A bacterial strain BL21/DE3 expressing T7 polymeraseGene from an inducible Lac promoter was used as a host.
34RNAi (RNA interference) and miRNAs 2001 Three labs published papers showing miRNA were generated by dicer.Explains generation of small temporal RNAs (stRNAs) such as lin-4 found in 1993.
35Screening for RNAi-deficient mutants The first mutants in the RNAi pathway identified by Tabara and Mello were called RNAi deficient (rde). These original screens were aimed at identifying of viable mutants, resistant to RNAi targeting pos-1, a gene important for viability.rde-1 nonrde-2 Ste/him/mutatorrde-3 Ste/him/mutatorrde-4 nonmut-2 Ste/him/mutatormut-7 Ste/him/mutatorExistence of related silencing pathways with distinct triggering mechanismsTabara H Cell, Vol. 99, 123–132,
36Existence of related silencing pathways with distinct triggering mechanisms
37Genetic links between RNAi and miRNA pathways. Rde-1 homologs. Grishok et al Cell, 106:Rde-1 homologs function:C. elegans RNAiDrosophila Development,epigenetic silencingArabidopsis development,gene silencingPhylogenetic Tree Grouping theRDE-1/AGO1/PIWI Protein Family members
38Function in miRNAs pathway Grishok et al Cell, 106:alg-1/alg-2 (lf) show defects that are similar to thoseobserved in heterochronic mutantsNothern blotPre-miRNAs processing is impairedFunction in miRNAs pathway
40RNAi pathway -post-transcriptional gene silencing On entering the cell, long dsRNAs act as a trigger of RNAi process.It is first processed by the RNAse III enzyme Dicer in an ATP-dependent reaction.Dicer processes dsRNAs into nt short interfering RNA (siRNA) with 2-nt 3' overhangs.The siRNAs are incorporated into the RNA-inducing silencing complex (RISC) which consists of an Argonaute (Ago) protein as one of its main components. Ago cleaves and discards the passenger (sense) strand of the siRNA duplex leading to activation of the RISC.The remaining guide (antisense) strand of the siRNA guides RISC to its homologous mRNA, resulting in the endonucleolytic cleavage of the target mRNA RNA-dependant-RNA-polymerase
41MicroRNAs - translational silencing. Novina C. & Sharp P Nature, 430: 161
42MicroRNAs – How many and what do they potentially regulate?
45Resources Alberts et al. Molecular Biology of the Cell. 4th Edition 21. Development of Multicellular Organisms. Caenorhabditis Elegans: Development from the Perspective of theIndividual Cell.ATLAS OF C. elegans ANATOMY. Chapter 1. INTRODUCTION TO C. elegans ANATOMY.miRNAsVella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook ,Ambros V. The functions of animal microRNAs. Nature Sep 16;431(7006): Review.Ambros V. The regulation of genes and genomes by small RNAs. Development. 2007;134(9):RNAiFire, A. et al (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391, 806–811.Timmons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854.Grishok. RNAi mechanisms in Caenorhabditis elegans.FEBS Lett Oct 31;579(26): Epub 2005 Aug 9. Review.Yigit E. et al. Analysis of the C. elegans Argonaute family reveals thatdistinct Argonautes act sequentially during RNAi.Cell Nov 17;127(4):
46Paper for the discussion : Parry DH, Xu J, Ruvkun G.A whole-genome RNAi Screen for C. elegans miRNA pathway genes.Curr Biol Dec 4;17(23):and Figure 1A from supplemental dataResourcesVella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook ,B.J. Reinhart, F.J. Slack, M. Basson, A.E. Pasquinelli, J.C. Bettinger, A.E. Rougvie, H.R. Horvitz and G. Ruvkun, The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans, Nature 403 (2000), pp. 901–906.Boutros M, Ahringer J. The art and design of genetic screens: RNA interference. Nat Rev Genet Jul;9(7):