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Lecture 9
• C. elegans cell biology
• C. elegans genetics
• C. elegans genome
Theoretically perfect model organism
• Well characterized life cycle-all stages easily
accessible.
• Well characterized genetic system.
• Well characterized genome-basically
sequenced and annotated.
• The ability to reintroduce DNA into the
organism-transgenesis.
• Closely related to humans-funding purposes.
Caenorabditis elegans
• Life cycle: short 3 1/2 days, transparent
organism, complete cell lineage known.
• Genetic system: both classical and RNAi
• Genome: first metazoan sequenced 1998
• Transgenesis: injection of DNA
• Related to humans?
Caenorabditis elegans
Hermaphrodite
Horvitz and Sternberg Nature 351, 535
Hermaphrodite and male
Wood 1998 The Nematode C. elegans
Cross section
tube within a tube
Wood 1998 The Nematode C. elegans
Wood 1998 The Nematode C. elegans
Life cycle
Wood 1998 The Nematode C. elegans
Life cycle
Hermaphrodite
558 nuclei
Males
560 nuclei
Wood 1998 The Nematode C. elegans
Hermaphrodite and male gonadogenesis
Wood 1998 The Nematode C. elegans
Hermaphrodite
959 somatic nuclei
Male
1,031 somatic nuclei
Life cycle
Wood 1998 The Nematode C. elegans
Worm’s brain
White et al. Phil. Trans. Royal Soc. London 314, 1-340
All neuronal connections known
White et al. Phil. Trans. Royal Soc. London 314, 1-340
Hermaphrodite and male
Wood 1998 The Nematode C. elegans
Fertilization and the first divisions
Kalthoff Analysis of Biological Development
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
Cell lineage
• Early divisions
• Lineage structure and nomenclature
• Cell death
• Repeated lineages
First four divisions and major blast cells
Wood 1998 The Nematode C. elegans
First four divisions and major blast cells
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
Wormbase
Temporal and spatial information
time
AB
AB.a
AB.p
M.vlpaa
Key blast cells are given upper case letters
The progeny are named by adding lower case
letters indicating the division axis:
a-anterior
p-posterior
d-dorsal
v-ventral
l-left
r-right
Following the lineage
M
M.v
M.vl
M.vlp
M.vlpa
M.vlpaa
great great great grandmoth
great great grandmother
great grandmother
grandmother
mother
daughter
Cell death
AB.alaaaala
l
alal
r
alar
Neuron in
DEAD
ring ganglion
Kalthoff Analysis of Biological Development
Repeated lineages
Wormbase
Repeated lineages
Wormbase
How is cell fate determined?
English vs American view
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
Fertilization and the first divisions
Kalthoff Analysis of Biological Development
How is cell fate determined?
English vs American view
Experimental approach: laser cell ablation
Nonautonomous determination
• Induction
• Equivalence groups
Induction
1
2
A cell or group of cells removed
from a second cell
that directs the developmental
fate of a second cell or group of
cells.
Example of induction
Anchor cell-gonad
signals
Epidermis
Vulva
Repeated lineages
Wormbase
Equivalence groups: Group of cells
that have equivalent pluripotent cell
fates.
Anchor cell/ Ventral uterine cell equivalence group
Individual A
Z1.ppp
AC
Z4.aaa
VU
Individual B
Z1.ppp
Z4.aaa
VU
AC
Anchor cell/ Ventral uterine cell equivalence group
Cell ablation experiment
Experiment A
Z1.ppp
AC
Z4.aaa
Experiment B
Z1.ppp
Z4.aaa
AC
Anchor cell/ Ventral uterine cell equivalence group
Cell ablation experiment
Experiment A
Z1.ppp
AC
Z4.aaa
Experiment B
Z1.ppp
Z4.aaa
AC
The remaining cell always becomes an AC.
The AC fate is the 1° (primary) cell fate.
Vulva equivalence group
Wormbase
Vulva equivalence group
P3.p
X
P8.p
X
Y
Z
Y
X
Vulva equivalence group
P3.p
P8.p
X
X
X
Y
X
Y
Y
Z
Z
Y
X
Z
Y
X
Y
X
Z
Vulva equivalence group
Z is the 1° cell fate
Y is the 2° cell fate
X is the 3° cell fate
C. elegans genetics
1. Self-fertilization
2. Systematic approach with RNAi
Self-fertilization and homozygousity
m/+
F0
m/m
m/+
+/+
F1
Self the population
m/m
F2
m/+
+/+
Mutagenesis and screens
P
0
young hermaphrodite
EMS
+/+ +/+ +/+ +/+ +/+ +/m +/+ +/+ ….. F1
self
All wild-type
self
F2
Males
X X hermaphrodite
X O male
At a frequency of 1/1000, males arise due to
nondisjunction of the X chromosome.
Complementation analysis
males m1/m1 X hermaphordites
m2/m2
Look at males only?
Complementation analysis
males m1/m1 X hermaphordites
m2/m2
1. All males have mutant phenotype
2. All males are wild-type
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Most
Wild-type
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Most
Wild-type
a- m+
a+ m-
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Most
Wild-type
a- m+
a+ m-
Some
a-
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Most
Wild-type
Some
a-
a- m+
a+ m-
a- m+
a- m+
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Rare
m-
Most
Wild-type
Some
a-
a- m+
a+ m-
a- m+
a- m+
Non complementation screen
EMS
male a+ m-/a+ m- X hermaphrodite a- m+/a- m+
Rare
ma- m-new
a+ m-
Most
Wild-type
Some
a-
a- m+
a+ m-
a- m+
a- m+
Transgenesis
YFG
rollD
Look for rolling progeny F1
Horvitz and Sternberg Nature 351, 535
Transgenesis
YFG
rollD
Look for rolling progeny F1
Look for rolling progeny in
F2
Horvitz and Sternberg Nature 351, 535
Transgenesis
Nucleus of F2 rolling progeny
YFG rollD YFG rollD rollD YFG rollD
Large concatenated
arrays that are stablely
maintained.
NCBI
RNAi inhibition of gene expression
1. RNAi discovered in C. elegans and plants.
2. Double stranded RNA results in the
degradation of homologous mRNA.
3. Double stranded RNA can be fed to
worms in the E. coli they eat.
4. Allows for the systematic inhibition of all
20,000 genes of C. elegans.
Systematic RNAi screens in C. elegans
Tuschl Nature 421, 220