16: Lecture Model organisms: C. elegans
Some facts about C. elegans
3 days life cycle
Invariant cell lineage
959 cells in hermaphrodite
1031 cells in male
Small genome 97 MB
19,000 genes (1 gene per 5 kb)
Two sexes: hermaphrodite and male
Six pairs of chromosomes
Read: reference C (uploaded in ELMS under course materials)
Fig. C.1
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Sex of C. elegans
XX: hermaphrodite
XO: male
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Fig. C.5b
Life cycle
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DNA transformation
Inject DNA into distal
syncytial gonad of
hermaphrodites
Irradiation promotes
integration of transgenes
into genome
Reporter constructs show
transgenes
Fig.C.8
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Fig. C.7b
Fig. C.5a
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Fig. C.7a
Invariant cell lineages
(959 somatic cells in hermaphrodite)
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2002 Nobel Prize Winners
For establishing C. elegans as the model organism
and the discovery of program cell death genes
Sydney Brenner
John Sulston
Bob Horvits
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C. elegans plays an important role in the discovery of
programmed cell death (apoptosis) genes
1. Apoptosis is important in development
Apoptosis patterns in developing Xenopus embryos detected via TUNEL
2. How to assay apoptosis?
TUNEL assay: TdT-mediated dUTP digoxigenin nick end labeling
Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase distinct from
other DNA polymerases in that it can add nucleotides to the 3 end of gene
segments without the use of a template strand.
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Functions of apoptosis
Sculpt body structures, e.g. hand digit
Serve some function but no longer needed
e.g. tadpole tail of frog.
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Apoptosis is suicide not murder
Cytoplasm shrinks
Chromosomes condense and fragment
Nuclear membrane breaks down
Apoptotic body formation
Engulfment of the cell corpse
video
Cell Death Can Be Studied at a Single Cell
Resolution
P11
X
X
P11aap
Adapted from Sulston and Horvitz,
Dev. Bology 56, 110-150, 1977
In 1980, E. Hedgecock isolated two cell death mutants
(ced-1 and ced-2) which are pivotal for identification
of the other cell death genes.
Wild-type
ced-1
Suppressor screens: ced-3 and ced-4
H. Ellis and R.H. Horvitz
Fig. C.15
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Bob Horvitz
ProgrammedCellDeathNaturallyoccurring,orprogrammed,cell
death(apoptosis)iscommonduringanimaldevelopment,and
abnormalitiesinprogrammedcelldeathareassociatedwithmany
humandiseases,includingcertaincancersandneurodegenerative
disorders.Ourlaboratoryhasdefinedamoleculargeneticpathwayfor
programmedcelldeath.Wehavecharacterizedgenesthatcausecells
todie,thatprotectcellsfromdying,thatfunctionintheengulfmentof
dyingcellsbytheirneighbors,andthatareinvolvedindestroyingthe
debrisgeneratedbycellcorpses.Mostofthesegeneshavehuman
counterparts.Forexample,thekillergene ced3isfacilitatedbyced4,
whichissimilartohumanAPAF1,identifiedbecauseitpromotes
caspaseactivationinabiochemicalsystem.Thefunctionof ced3encodesacaspase
(cysteineaspartateprotease);mammaliancaspasessimilarlycause
programmedcelldeath.Theactionof ced4is
blockedbyced9,whichprotectscellsagainstprogrammedcelldeath
andissimilartothehumanprotooncogene BCL2,whichalsoprotects
againstcelldeath.Theactivityof ced9isinhibitedbythewormkiller
geneegl1,whichissimilartoanumberofmammaliankillergenes.
Theactivityofegl1iscontrolledinacellspecificfashionbygenes
thatspecifywhichcellsaretoliveandwhicharetodie
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Figure 13.48. Regulators and effectors of apoptosis Many cell death signals induce
apoptosis via a conserved pathway of regulators, adaptors, and caspases. In C. elegans, the
negative regulator Ced-9 inhibits apoptosis by binding to the adaptor Ced-4. In the absence of
inhibition by Ced-9, Ced-4 binds two molecules of the caspase Ced-3, resulting in
autocleavage and caspase activation. In mammals, regulators of the Bcl-2 family (Ced-9
homologs) act at the mitochondria to control release of cytochrome c, which is required for
the binding of caspase-9 to the adaptor Apaf-1 (the Ced-4 homolog). Release of cytochrome c
from mitochondria thus signals the activation of caspase-9, which then activates downstream
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caspases to induce apoptosis
RNAi was first discovered in C. elegans
Nobel prize-2006
Andy Fire
Fire et al., (1998) Potent and specific genetic interference by
double stranded RNA in C. elegans. Nature vol 391, 806-810
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mex3 mRNA expression by in situ hybridization
WT without probe
Antisense RNA
WT with probe
dsRNA
Fire et al., (1998) Potent and specific genetic interference by
double stranded RNA in C. elegans. Nature vol 391, 806-810
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RNAi
Fig. C.9
The C. elegans Lin-4 was the first microRNA discovered
In Dr. Victor Ambros lab
Lee RC, Feinbaum RL, Ambros V.
(1993) The C. elegans heterochronic gene
lin-4 encodes small RNAs with antisense complementarity to lin-14.
Cell 75(5):843-54.
Lee RC, Ambros V. (2001) An extensive class of small RNAs in Caenorhabditis elegans.
Science. 2001 Oct 26;294(5543):862-4.
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Genetic pathway that regulates developmental timing
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lin-4
lin-4
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siRNA vs. miRNA
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A good movie on RNAi
http://www.nature.com/focus/rnai/animations/index.html
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