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.
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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”
1991-98 RNAi and miRNA discovered in worms. (Nobel Prize: Fire,
Mello 2006)
1994 - 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.
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.
C. elegans morphology
Organs and tissues
Muscles
http://www.wormatlas.org
Epidermal system
Digestive system
Reproductive system
Muscular system
Excretory system
Nervous system
Nervous system
Hermaphrodite 302 neurons (118 types)
5000 chemical synapses
2000 neuromuscular
junctions
Male
381 neurons
By comparison
105 neurons in flys, ~300 in worms
Interneurons and motorneurons
Major neurotransmitters:
Ach -exitatory
GABA- inhibitory
Serotonin (HSN neuron- egg laying)
Dopamine
FMRF amide peptides
Wood W.B. 1988. The Nematode Caenorhabditis elegans. 1-667.
Life cycle of Caenorhabditis elegans
Proliferation
 generation of embryonic founder cells
 bulk of cell divisions and gastrulation.
a spheroid of cells:
ectoderm - hypodermis and neurons
mesoderm - pharynx and muscle,
endoderm - germline and intestine.
Organogenesis
terminal differentiation
3 days at 22C
http://www.wormatlas.org
C. elegans cell lineage
• One amazing advantage of worms is that every
worm has the exact same number of somatic
cells!
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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.
The divisions are invariant in pattern, timing, and
orientation of each division.
Here it is: C. elegans cell lineage
EMS
Know how to read a lineage diagram!
1) Each branch indicates a cell division
2) When a cell differentiates and no longer divides, line ends
3) Line ending in X= programmed cell death
4) Moving from top to bottom is moving ahead in time
P1
P2 P
3
Postembryonic 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.
Divisions
Migrations
Differentiation
Death
http://www.wormatlas.org
Direct 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 56:110-156 (1977)
Founder cell derivation by asymmetric cell
division and inductions
http://www.wormclassroom.org/db/sampleLineage.html
Founder cells: AB, MS, E, C, D and P4.
Distinctive properties of founder cells defined by:
 division rate
 nature of their progeny
Germline formation in C. elegans
6 Founder Cells
+98 die
+14 die
Small posterior cell goes
germline
+1 dies
How are invariant lineages
established and maintained?
• Partitioning of prexisting
maternal components
• Cell-autonomous programs
• Cell-cell interactions
Asymmetric cell division
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Sperm entry defines posterior pole. Sperm aster microtubules organize microfilaments
needed to establish asymmetries.
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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.
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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.
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. division.
Where do specific tissues come from?
Lineages do not strictly produce single tissue types.
AB
MS
E
C
D
P4
While 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.
Cell death can be a genetically programmed cell fate.
Indicate death event
The Nematode Caenorhabditis Elegans.
1988. Editor William B. Wood ,
Cold Spring Harbor Laboratory.
 Specific cells with diverse developmental origins undergo programmed cell death at specific
times 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.
Genetics of Programmed Cell Death:
CED-3, CED-4 and CED-9 have human counterparts.
A ced-1 has defect in the engulfment of dying
cells. Programmed cell death does not occur in
a ced-3 mutant. ced-3 (caspase) is a killer.
egl-1 (dead HSN neuron)
Search for supression
of egl (HSN restored)
ced-4 (Apaf-1-like-protein) 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.org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.html
The genetic pathway for programmed
cell death in C. elegans
http://nobelprize.org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.html
The principle of biological universality
 “One point that emerges from the studies of programmed
cell death in C. 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,”

and it underlies my strong conviction that the rigorous,
detailed and analytic study of the biology of any organism is
likely to lead to findings of importance in the understanding
of other organisms.”
http://nobelprize.org/nobel_prizes/medicine/laureates/2002/horvitz-lecture.html
miRNAs
• miRNAs were discovered in C. elegans.
• Additionally, much of what we know about miRNAs
and 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 of
miRNAs, and their similarity to siRNAs, has made a
huge impact on our thinking about genetic control.
Genes that affect timing and Discovery of
microRNAs (miRNAs)
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The definition of heterochrony is a change
in the relative timing of developmental events. Each
scale of development, the cycle of cell divisions, the growth
of tissues, the formation of organs, requires proper timing.
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Two general phenotypes are seen in heterochronic
mutants —
 ‘precocious,’ in which developmental events are skipped,
 ‘retarded,’ in which they are repeated.
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A heterochronic mutation may affect different tissues:
intestine, epidermis, muscle, and neurons, and
different kinds of developmental events:
a pattern of cell division, a cell cycle lengths, and
differentiation.
An example: lin-4 and lin-14 mutants
Hypodermal cell lineage
Moss E. 2007. Current Biology, R425.
Lin-4 regulates transition from L1 to L2 stage.
Lin-14 gene is a regulator of transcription.
Lin-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 complementary
One of lin-4’s target genes, lin-14,
sequences on target messenger
encodes a novel nuclear protein and is
a putative transcription factor. The lin4 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. 2004. Nature 431, 350-355
The 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 L4to-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. 2007. Current Biology, R425.
Temporal 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/10.1895/
wormbook.1.26.1, http://www.wormbook.org.
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.
MicroRNA functions in animals and disease.
E. Wienholds, R.H.A. Plasterk / FEBS Letters 579 (2005) 5911–5922
miRNA-mediated repression of translation
Nelson T. 2007. 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.
RNAi (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!!!!!
• 1991-1997 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 RNAi
• Essential for transposon silencing.
• 2000 Dicer enzyme cleaves dsRNA precursors to 21-23bp duplex
oligonuc.
Important characteristics of RNAi in C. elegans
 systemic
 amplified
 heritable
Ways to introduce dsRNA into worms:
 Injection
 Soaking
 feeding
Plasmids for feeding
Timmins L. & Fire A. 1998. Nature,
395:854.
Segments were cloned between flanking copies of
bacteriophage T7 promoter into a bacterial plasmid vector.
A bacterial strain BL21/DE3 expressing T7 polymerase
Gene from an inducible Lac promoter was used as a host.
Large-scale screens for
RNAi deficient mutants.
Production of genome
wide collections of RNAi
feeding constructs
RNAi (RNA interference)
RNAi-induced silencing complex
RNAi (RNA interference)
and miRNAs
1. 2001 Three labs published
papers showing miRNA were
generated by dicer.
2. Explains generation of small
temporal RNAs (stRNAs)
such as lin-4 found in 1993.
Screening 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 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. 1999. Cell, Vol. 99, 123–132,
Existence of related
silencing pathways with
distinct triggering
mechanisms
Existence of related silencing pathways with distinct
triggering mechanisms
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/mello-lecture.html
Genetic links between RNAi and miRNA pathways.
Rde-1 homologs.
Grishok et al. 2001. Cell, 106: 23-34.
Rde-1 homologs function:
C. elegans
- RNAi
Drosophila - Development,
epigenetic silencing
Arabidopsis - development,
gene silencing
Phylogenetic Tree Grouping the
RDE-1/AGO1/PIWI Protein Family
members
Grishok et al. 2001. Cell, 106: 23-34.
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
Link between silencing pathways
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/mello-lecture.html
RNAi 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 21-23
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.
RNA-dependant-RNA-polymerase
The remaining guide (antisense)
strand of the siRNA guides RISC to
its homologous mRNA, resulting in
the endonucleolytic cleavage of the
target mRNA
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/mello-lecture.html
MicroRNAs - translational silencing.
Novina C. & Sharp P. 2004. Nature, 430: 161
MicroRNAs – How many and what do
they potentially regulate?
Resources
Alberts et al. Molecular Biology of the Cell. 4th Edition
21. Development of Multicellular Organisms. Caenorhabditis Elegans: Development from the Perspective of the
Individual Cell.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=Perspective,Individual,Development,Cell,
ATLAS OF C. elegans ANATOMY. Chapter 1. INTRODUCTION TO C. elegans ANATOMY.
http://www.wormatlas.org/
miRNAs
Vella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),
WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/
wormbook.1.26.1, http://www.wormbook.org.
Ambros V. The functions of animal microRNAs. Nature. 2004 Sep 16;431(7006):350-5. Review.
Ambros V. The regulation of genes and genomes by small RNAs. Development. 2007;134(9):1635-41.
RNAi
Fire, 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. 2005 Oct 31;579(26):5932-9. Epub 2005 Aug 9. Review.
Yigit E. et al. Analysis of the C. elegans Argonaute family reveals that
distinct Argonautes act sequentially during RNAi.
Cell. 2006 Nov 17;127(4):747-57.
Paper for the discussion :
Parry DH, Xu J, Ruvkun G.
A whole-genome RNAi Screen for C. elegans miRNA pathway genes.
Curr Biol. 2007 Dec 4;17(23):2013-22.
and Figure 1A from supplemental data
Resources
Vella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),
WormBook, ed. The C. elegans Research Community, WormBook,
doi/10.1895/
wormbook.1.26.1, http://www.wormbook.org.
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. 2008 Jul;9(7):554-66.