C. elegans cell biology

1
Lecture 9

• C. elegans cell biology
• C. elegans genetics
• C. elegans genome

2
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.

3
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?

4
Caenorabditis elegans
Hermaphrodite
Horvitz and Sternberg Nature 351, 535
5
Hermaphrodite and male
Wood 1998 The Nematode C. elegans
6
Cross section tube within a tube
Wood 1998 The Nematode C. elegans
7
Wood 1998 The Nematode C. elegans
8
Life cycle
Wood 1998 The Nematode C. elegans
9
Life cycle
Hermaphrodite 558 nuclei Males 560 nuclei
Wood 1998 The Nematode C. elegans
10
Hermaphrodite and male gonadogenesis
Wood 1998 The Nematode C. elegans
11
Life cycle
Hermaphrodite 959 somatic nuclei Male 1,031
somatic nuclei
Wood 1998 The Nematode C. elegans
12
Worms brain
White et al. Phil. Trans. Royal Soc. London 314,
1-340
13
All neuronal connections known
White et al. Phil. Trans. Royal Soc. London 314,
1-340
14
Hermaphrodite and male
Wood 1998 The Nematode C. elegans
15
Fertilization and the first divisions
Kalthoff Analysis of Biological Development
16
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
17
Cell lineage

• Early divisions
• Lineage structure and nomenclature
• Cell death
• Repeated lineages

18
First four divisions and major blast cells
Wood 1998 The Nematode C. elegans
19
First four divisions and major blast cells
20
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
21
Wormbase
22
Temporal and spatial information
time
AB
AB.a
AB.p
23
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
24
Following the lineage
M great great great grandmoth M.v great great
grandmother M.vl great grandmother M.vlp grandmo
ther M.vlpa mother M.vlpaa daughter
25
Cell death
AB.alaaaala
l
r
alal
alar
Neuron in ring ganglion
DEAD
Kalthoff Analysis of Biological Development
26
Repeated lineages
Wormbase
27
Repeated lineages
Wormbase
28
How is cell fate determined?
English vs American view
29
Complete cell lineage
Slack and Ruvkun Annu. Rev, Genet. 31, 611
30
Fertilization and the first divisions
Kalthoff Analysis of Biological Development
31
How is cell fate determined?
English vs American view
Experimental approach laser cell ablation
32
Nonautonomous determination

• Induction
• Equivalence groups

33
Induction
A cell or group of cells removed from a second
cell
1
that directs the developmental fate of a second
cell or group of cells.
2
34
Example of induction
Anchor cell-gonad
signals
Epidermis
Vulva
35
Repeated lineages
Wormbase
36
Equivalence groups Group of cells that have
equivalent pluripotent cell fates.
37
Anchor cell/ Ventral uterine cell equivalence
group
Individual A
Individual B
Z1.ppp
Z1.ppp
Z4.aaa
Z4.aaa
AC
AC
VU
VU
38
Anchor cell/ Ventral uterine cell equivalence
group
Cell ablation experiment
Experiment A
Experiment B
Z1.ppp
Z1.ppp
Z4.aaa
Z4.aaa
AC
AC
39
Anchor cell/ Ventral uterine cell equivalence
group
Cell ablation experiment
Experiment A
Experiment B
Z1.ppp
Z1.ppp
Z4.aaa
Z4.aaa
AC
AC
The remaining cell always becomes an AC. The AC
fate is the 1 (primary) cell fate.
40
Vulva equivalence group
Wormbase
41
Vulva equivalence group
P3.p
P8.p
X
Y
Z
Y
X
X
42
Vulva equivalence group
P3.p
P8.p
X
Y
Z
Y
X
X
X
Y
Z
X
Y
X
Y
X
Y
Z
Z
43
Vulva equivalence group
Z is the 1 cell fate Y is the 2 cell fate X is
the 3 cell fate
44
C. elegans genetics
1. Self-fertilization 2. Systematic approach
with RNAi
45
Self-fertilization and homozygousity
m/ F0
m/m m/ / F1
Self the population
m/m m/ / F2
46
Mutagenesis and screens
P0
young hermaphrodite
EMS
/ / / / / /m / / ..
F1
self
self
F2
All wild-type
47
Males
X X hermaphrodite X O male
At a frequency of 1/1000, males arise due to
nondisjunction of the X chromosome.
48
Complementation analysis
males m1/m1 X hermaphordites m2/m2
Look at males only?
49
Complementation analysis
males m1/m1 X hermaphordites m2/m2
1. All males have mutant phenotype 2. All males
are wild-type
50
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Most Wild-type
51
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Most Wild-type
a- m a m-
52
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Some a-
Most Wild-type
a- m a m-
53
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Some a-
Most Wild-type
a- m a- m
a- m a m-
54
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Some a-
Most Wild-type
Rare m-
a- m a- m
a- m a m-
55
Non complementation screen
EMS
male a m-/a m- X hermaphrodite a- m/a- m
Some a-
Most Wild-type
Rare m-
a- m a- m
a- m-new a m-
a- m a m-
56
Transgenesis
YFG
rollD
Look for rolling progeny F1
Horvitz and Sternberg Nature 351, 535
57
Transgenesis
YFG
rollD
Look for rolling progeny F1
Look for rolling progeny in F2
Horvitz and Sternberg Nature 351, 535
58
Transgenesis
Nucleus of F2 rolling progeny
YFG
rollD
YFG
YFG
rollD
rollD
rollD
Large concatenated arrays that are stablely
maintained.
59
NCBI
60
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.
61
Systematic RNAi screens in C. elegans
Tuschl Nature 421, 220