C.

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.

C. elegans timeline.
• • • • • Developed by Sydney Brenner (1963) Mutants published by Brenner (1974) 1976 postembryonic cell lineages determined (Sulson and Horwitz) 1983 complete embryonic cell lineages determined (Deppe et al., Sulson et al.) 1982 "Programmed cell death" (Horwitz et al.) Nobel Prize Brenner, Sulson, Horwitz 2002 1986 Complete connectivity of nervous system established (White et al.) "The mind of a worm” 1991-98 RNAi and miRNA discovered in worms. Nobel Prize: Fire, Mello 2006 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 17,500 genes, about 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.

C. (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 agar plates with lawn of bacteria. • They have a short generation time- 3 days from egg-laying to adulthood, brood size > 300. • They are transparent, so internal anatomy can be easily observed.
Goldstein lab
http://www.bio.unc.edu/faculty/goldstein/lab/crawl.mov

Caenorhabditis elegans
959 somatic cells
XX karyotype

XO karyotype 1031 cells
http://www.wormatlas.org

• 5 autosomes I-V and X (6 total) • XX animals become hermaphrodites • XO animals become males (chromosome non-disjunction) • With one less chromosome, males develop 79 more neurons and 25 more muscle cells than hermaphrodites, used for mating. • Hermaphrodite produces many progeny--300 worms! • Adult hermaphrodites have 959 somatic cells while males have 1031.

org . Elegans morphology Organs and tissues Muscles Epidermal system Digestive system Reproductive system Muscular system Excretory system Nervous system http://www.C.wormatlas.

. The Nematode Caenorhabditis elegans. ~300 in worms Interneurons and motorneurons Major neurotransmitters: Ach -exitatory GABA.inhibitory Serotonine (HSN neuron. 1988.B. 1-667.Nervous system Hermaphrodite 302 neurons 5000 chemical synapses 2000 neuromuscular junctions Male 381 neurones By comparison 105 neurons in flys.egg laying) Dopamine FMRF amide peptides Wood W.

hypodermis and neurons. Organogenesis terminal differentiation 3 days at 22C http://www. a spheroid of cells: ectoderm .Life cycle of Caenorhabditis elegans Proliferation  generation of embryonic founder cells  bulk of cell divisions and gastrulation.pharynx and muscle. endoderm .org .wormatlas. mesoderm .germline and intestine.

. The divisions are invariant in pattern. elegans cell lineage • The most important and incredible advantage of worms is that every worm has the exact same number of somatic cells!    Hatching larva=558 cells Males (XO)=1031 somatic cells+ ~1000 sperm Hermaphrodite (XX)= 959 somatic cells+ ~2000 eggs and sperm. • Somatic cells arise by an INVARIANT cell lineage. and orientation of each division. timing.C.

Here it is: C. line ends 3) Line ending in X= programmed cell death 4) Moving from top to bottom is moving ahead in time . elegans cell lineage EMS P1 P2 P 3 Know how to read a lineage diagram! 1) Each branch indicates a cell division 2) When a cell differentiates and no longer divides.

a first stage larva has approximately 671 cells. 113 undergo programmed death in the course of development. Divisions Migrations Differentiation Death http://www. In the presence of food.wormatlas. About 10% of the remaining 558 cells in a newly hatched larva (51 in hermaphrodites. 55 in the male).org . are blast cells that divide further. cell divisions resume and postembryonic developmental program begins 3 hours after hatching.Postembryonic cell lineage was completed first.

P10 metaphase. Developmental Biology 56:110-156 (1977) . 26 min 10 anaphase. P9 metaphase. P9 prophase. 29 min. P10 telophase.Direct observations are possible Cell divisions. 24 min. Sulston & Horvitz. Sequential photographs of an L1 hermaphrodite. 16 and 21 min. 33 and 34 min. interphase. P10 prophase. 0 min. lateral view. 27 mm. ventral cord neurons. Nomarski optics vcn.

wormclassroom.Founder cell derivation by asymmetric cell division and inductions http://www.org/db/sampleLineage. C. Distinctive properties of founder cells defined by:  division rate  general nature of there progeny . D and P4.html Founder cells: AB. MS. E.

How are invariant lineages established and maintained? • Partitioning of prexisting maternal components • Cell-autonomous programs • Cell-cell interactions .

just one cell. PKC-3 associate with anterior surface. P granules(ribonucleoprotein particals) segregate to posterior pole until 16-cell stage and P4. Several genes identified that are required for this partitioning of material and are called par genes in worms. PAR-3. Sperm aster microtubules organize microfilaments needed to establish asymmetries. A-P partitioning occurs in all germline precursors. -2 associate with posterior surface. will give a rise to germline. • • .Asymmetric cell division • Sperm entry defines posterior pole. -6. They coordinate the polarization of cytoskeleton affecting distribution of other cell components. Differential partitioning of maternally expressed proteins to anterior and posterior of egg occur: PAR-1.

division.Early inductions P2-EMS • 1st transcription occurs at 4-cell stage in EMS. • Can block transcription get to 100 cell stage • Lineage commitment can occur through multiple mechanisms • P2 induces ABp but not ABa by contact • P2 induces EMS asymm. .

the muscle. nervous system & skin cells each arise from multiple lineages. that muscles do not have one “founder” cell early in development. for example.Where do specific tissues come from? Lineages do not strictly produce single tissue types. This means. . AB MS E C D P4 While intestine & germ-line come from single founder cells.

Cell death can be a genetically programmed cell fate.  Programmed cell death is characterized by a series of specific morphological changes.  Specific cells with diverse developmental origins undergo programmed cell death at specific times during development. Indicate death event The Nematode Caenorhabditis Elegans.  There must be genes that control both the decision to express that fate and the execution. Editor William B. Cold Spring Harbor Laboratory. Wood . . 1988.

html . CED-4 and CED-9 have human counterparts.org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.Genetics of Programmed Cell Death: CED-3. egl-1 (dead HSN neuron) Search for supression of egl (HSN restored) ced-4 (Apaf-1-like-protein) is a killer. Programmed cell death does not occur in a ced-3 mutant. ced-3 (caspase) is a killer. The sister of NSM neuron in the pharynx survives instead of dying ced-9 (gf) ced-9 has similarities to the proto-oncogene Bcl-2 (B cell lymphoma) http://nobelprize. A ced-1 has defect in the engulfment of dying cells.

elegans. http://nobelprize.The overall molecular genetic pathway for programmed cell death in C.html .org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.

detailed and analytic study of the biology of any organism is likely to lead to findings of importance in the understanding of other organisms.The principle of biological universality.  “One point that emerges from the studies of programmed cell death in C.”  and it underlies my strong conviction that the rigorous...org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.” http://nobelprize. elegans and other organisms is the striking similarity of genes and gene pathways among organisms that are as superficially distinct as worms and humans. I like to refer to this theme as “The principle of biological universality.html .

miRNAs • miRNAs were discovered in C. elegans has over 100 miRNAs. . and their similarity to siRNAs. • Most biological processes are touched in some way by miRNAs. • C. much of what we know about miRNAs and their targets has come from studies in C. • Additionally. • The excitement generated by the discovery of miRNAs. elegans. elegans. Humans have over 200. has made a huge impact on our thinking about genetic control.

epidermis.Genes that affect timing and Discovery of microRNAs (miRNAs). and differentiation.   A heterochronic mutation may affect different tissues: intestine. the cycle of cell divisions. and different kinds of developmental events: a pattern of cell division.  Two general phenotypes are seen in heterochronic mutants —  ‘precocious.’ in which developmental events are skipped. the growth of tissues. the formation of organs. requires proper timing. muscle. .  ‘retarded. and neurons. a cell cycle lengths. Each scale of development.’ in which they are repeated.  The definition of heterochrony is a change in the relative timing of developmental events.

An example: lin-4 and lin-14 mutants Hypodermal cell lineage Moss E. Lin-14 gene is a regulator of transcription. Current Biology. . 2007. Lin-4 regulates transition from L1 to L2 stage. R425.

lin-14. sequences on target messenger encodes a novel nuclear protein and is a putative transcription factor. it is of no consequence. 350-355 . imperfectly base-paired to complementary Ambros V. (Posttranscriptional control). 2004. lin-4 encodes a small RNA molecule. These hairpin precursor is a characteristic feature of the miRNA class of regulatory RNAs. Upon lin-4 expression. One of lin-4’s target genes. Nature 431. Although transcription from the lin-14 gene still occurs. The lin4 microRNA regulates lin-14 through specific sequences in the 3’ UTR of the lin-14 mRNA. a rare 61 nt pre-lin-4 precursor that is processed into a 22nt miRNA.Lin-4 is the first microRNA gene. lin-14 protein levels are reduced.

and die by bursting through the vulva (Reinhart et al. lin-41 is one of the targets.. Current Biology. Moss E. let-7 mutants fail to execute the L4-to-adult transition (the seam cells fail to terminally differentiate and continue to divide). elegans. . 2000). elegans.The conservation of let-7 across animal phylogeny showed that microRNAs are not exclusive to C. 2007. let-7 controls the L4-to-adult transition in C. R425.

C.C. WormBook. MicroRNAs increase in abundance at each stage and repress specific targets that encode developmental regulators. 2005).J.1895/ wormbook. http://www. .org. and Slack. M.1. F.1. Vella.wormbook.26. The change in regulators at each stage leads to a succession of developmental events. doi/10. ed. WormBook. elegans microRNAs (September 21. elegans Research Community. The C.Temporal regulation of the miRNAs lin-4 and let-7 and their target genes.

R. E.MicroRNA functions in animals and disease. Plasterk / FEBS Letters 579 (2005) 5911–5922 .A. Wienholds.H.

In the case of post-initiation repression – which includes premature ribosome drop off. which might result in degradation (increased turnover). through sequence complementarity. 3) targeted mRNAs could be sequestered from the translational machinery and degraded or stored for subsequent use. including promoting deadenylation. It might take place in P bodies (denoted by P). or through inhibition of translation post-initiation. 2) miRNPs can have other effects on targeted mRNAs. 2007. 23: 243. slowed or stalled elongation. This interaction can have direct and indirect effects on translation: 1) Direct effects through inhibition of initiation of translation. . Target mRNAs are recognized by miRNAs in the form of ribonucleoprotein complexes (miRNPs). which results in prevention of ribosome association with the target mRNA.miRNA-mediated repression of translation Nelson T. and cotranslational protein degradation – the repressed mRNA seems to be present in polysomes. TRENDS in Genetics. which are enriched for factors involved in mRNA degradation.

enhanced degradation of target transcript. . • 1998 Effect is post-transcriptional.1995) • 1998 Fire et al. showed that antisense RNA could inhibit gene expression when injected into worms. • 1999 First genes identified for RNAi • Essential for transposon silencing. • 2000 Dicer enzyme cleaves dsRNA precursors to 21-23bp duplex oligonuc. causes sequence-specific degradation of homologous mRNA sequences. when introduced into a cell. • 1991 Fire et al. and the Mello lab showed that combination of sense and antisense worked all the time on certain genes. • Didn't always work!!!!! • 1991-1997 several papers showed that either antisense or sense RNA could interfere (Guo and Kemphues Cell:81. Probably got hairpins with sense or antisense alone.RNAi (RNA interference) RNAi is an evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA).

elegans  systemic  amplified  heritable Ways to introduce dsRNA into worms:  Injection  Soaking  feeding .Important characteristics of RNAi in C.

Large-scale screens for RNAi deficient mutants. 395:854.Plasmids for feeding Timmins L. 1998. & Fire A. Production of genome wide collections of RNAi feeding constructs . A bacterial strain BL21/DE3 expressing T7 polymerase Gene from an inducible Lac promoter was used as a host. Segments were cloned between flanking copies of bacteriophage T7 promoter into a bacterial plasmid vector. Nature.

RNAi (RNA interference) RNAi-induced silencing complex .

2. . 2001 Three labs published papers showing miRNA were generated by dicer.RNAi (RNA interference) and miRNAs 1. Explains generation of small temporal RNAs (stRNAs) such as lin-4 found in 1993.

1999. a gene important for viability. Existence of related silencing pathways with distinct triggering mechanisms . Vol. 99.Screening for RNAi-deficient mutants The first mutants in the RNAi pathway identified by Tabara and Mello were called RNAi deficient (rde). rde-1 non rde-2 Ste/him/mutator rde-3 Ste/him/mutator rde-4 non mut-2 Ste/him/mutator mut-7 Ste/him/mutator Tabara H. 123–132. resistant to RNAi targeting pos-1. Cell. These original screens were aimed at identifying of viable mutants.

Existence of related silencing pathways with distinct triggering mechanisms http://nobelprize.html .org/nobel_prizes/medicine/laureates/2006/mello-lecture.

RNAi Drosophila . 106: 23-34. Rde-1 homologs. Cell. 2001.development. epigenetic silencing Arabidopsis .Genetic links between RNAi and miRNA pathways. gene silencing Phylogenetic Tree Grouping the RDE-1/AGO1/PIWI Protein Family members . elegans . Grishok et al. Rde-1 homologs function: C.Development.

alg-1/alg-2 (lf) show defects that are similar to those observed in heterochronic mutants Nothern blot Pre-miRNAs processing is impaired Function in miRNAs pathway . Cell.Grishok et al. 2001. 106: 23-34.

Link between silencing pathways http://nobelprize.html .org/nobel_prizes/medicine/laureates/2006/mello-lecture.

It is first processed by the RNAse III enzyme Dicer in an ATP-dependent reaction.RNAi pathway -post-transcriptional gene silencing On entering the cell.org/nobel_prizes/medicine/laureates/2006/mello-lecture. The siRNAs are incorporated into the RNAinducing silencing complex (RISC) which RNA-dependant-RNA-polymerase consists of an Argonaute (Ago) protein as one of its main components.html . Dicer processes dsRNAs into 21-23 nt short interfering RNA (siRNA) with 2-nt 3' overhangs. The remaining guide (antisense) strand of the siRNA guides RISC to its homologous mRNA. long dsRNAs act as a trigger of RNAi process. resulting in the endonucleolytic cleavage of the target mRNA http://nobelprize. Ago cleaves and discards the passenger (sense) strand of the siRNA duplex leading to activation of the RISC.

Nature. 2004.MicroRNAs . Novina C. 430: 161 .translational silencing. & Sharp P.

L. Nature 395. ATLAS OF C. M. WormBook.C. et al.ncbi. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 4th Edition 21. Alberts et al. Yigit E. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. . A. et al (1998). Grishok. Epub 2005 Aug 9. 2005 Oct 31.431(7006):350-5.wormatlas. Review. INTRODUCTION TO C. Ambros V.org.134(9):1635-41. A. RNAi mechanisms in Caenorhabditis elegans. Development of Multicellular Organisms. Analysis of the C. C.gov/books/bv. (1998).wormbook. and Fire. http://www. elegans microRNAs (September 21. Chapter 1.org/ miRNAs Vella. http://www.1. doi/10.Resources.. 806–811.nlm. RNAi Fire. Ambros V. Review. The C. FEBS Lett. 2004 Sep 16. elegans Research Community. Molecular Biology of the Cell. Nature 391.Individual. Timmons. 854. and Slack. 2005).579(26):5932-9. WormBook. Caenorhabditis Elegans: Development from the Perspective of the Individual Cell. elegans ANATOMY.1895/ wormbook. Cell.1. 2007. Specific interference by ingested dsRNA. F. The regulation of genes and genomes by small RNAs. Development.26.127(4):747-57. 2006 Nov 17. http://www. ed.J.nih.Cell.fcgi?highlight=Perspective.Development. elegans ANATOMY. The functions of animal microRNAs.

C. http://www. Rougvie. and Figure 1A from supplemental data Resources Vella. B. A. . Ahringer J. elegans Research Community. Slack. and Slack. WormBook. 2005).17(23):2013-22.E.R. Ruvkun. elegans miRNA pathway genes. Xu J.26.J. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Boutros M. Ruvkun G. The C. The art and design of genetic screens: RNA interference. Curr Biol. Reinhart. elegans microRNAs (September 21. Bettinger.J. Horvitz and G. Pasquinelli. 2008 Jul. C. A whole-genome RNAi Screen for C. F. pp. Basson. 2007 Dec 4.J. 901– 906.wormbook.E.1.org. F. Nat Rev Genet. ed.C. M. A. doi/10.1. Nature 403 (2000).Paper for the discussion : Parry DH. J. H.9(7):554-66.1895/ wormbook. M. WormBook.

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