The invariant lineage of C.

elegans

1090 cells
But what about 959?

cell death!
Sulston, J.E. and H.R. Horvitz (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.
Dev. Biol. 56(1), 110-156.
http://www.sciencedirect.com/science/article/pii/0012160677901580
Sulston, J.E. et al. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans.
Dev. Biol. 100(1), 64-119.
http://www.sciencedirect.com/science/article/pii/0012160683902014
 Apoptosis in C. elegans
•  Exactly 131 of the 1090 somatic cells are eliminated through cell death
–  113 of these cell deaths occur during embryogenesis
–  the other 18 occur during larval development The pattern of cell death is highly
reproducible in C. elegans

Altun, Z.F. and Hall, D.H. (2009). Introduction. In WormAtlas. doi:10.3908/wormatlas.1.1

Apoptosis in C. elegans
 Apoptosis in C. elegans

•  Lose contact with neighboring cells

•  Round-up and condense
•  Nuclei become pyknotic
•  Engulfment
•  Degradation Image courtesy of Nadin Memar

Within 30-40 minutes, cells undergo the typical

morphological changes, are engulfed and degraded.
 Apoptosis in C. elegans
1970 1980 1990 2000

Bcl-2

In no other model
organism do you
find such an
invariant pattern
of cell death

Why did C. elegans turn out to be very important for studies on apoptosis?
Making mutants
 Making mutants

•  Spontaneous mutations occur naturally in the DNA of all living organisms

…but don’t occur frequently enough for the purpose of forward genetic screens

We need more
mutations!
 Making mutants

•  Spontaneous mutations occur naturally in the DNA of all living organisms

…but don’t occur frequently enough for the purpose of forward genetic screens

Perfect!

EMS
Ethyl methanesulfonate
Making mutants

T A
A T
G C
C G
G C
A T
T A
C G
Making mutants

T A
A T EMS

G C
C G O-6-ethylguanine

G C
A T
T A
C G
Making mutants

A
T
C
G
C
T
A
G
 Making mutants

T A
A T
thymine
G C
is paired with T G
O-6-ethylguanine
G C
A T
T A
C G
Making mutants

T
A
G
T
G
A
T
C
 Mutagenesis
original

T A T A
A T A T
G C G C C:G
C G T A has become
T:A
G C G C
A T A T The exchange of a single nucleotide
T A T A pair is a point mutation

C G C G
 Mutagenesis
point mutation

•  Point mutations are small scale mutations small scale

•  Translocations, deletions, and duplications are large scale mutations

translocation

large scale
 Mutagenesis

Mutations might:

•  cause lower expression of the gene

•  make the gene product less stable
•  make the gene product non-functional

cis-regulatory elements promoter coding region cis-regulatory elements

loss-of-function

exon intron UTR promoter enhancer silencer

 Loss-of-function mutations
100%

%
of normal
gene
expression
0%
hypomorphic mutation

amorphic mutation wild-type

(null mutation)

Each loss-of-function can cause a specific mutant phenotype

 Loss-of-function mutations

•  In general, loss-of-function mutations are recessive

•  C. elegans hermaphrodites have 6 pairs of chromosomes
–  males only have a single X chromosome

I II III IV V X
 Recessive mutations

wild-type
allele

mutant
allele

Homozygous Heterozygous Homozygous

wild-type mutant

Normal phenotype Mutant

phenotype
 Recessive mutations

~90% of EMS-induced mutations are recessive

~10% are dominant

 Dominant mutations

wild-type
allele

mutant
allele

Homozygous Heterozygous Homozygous

wild-type mutant

Normal Mutant phenotype

phenotype
 Forward genetic screen

somatic cell germ cells

(oocyte and sperm)

diploid haploid
 Forward genetic screen
EMS

P0
+
+
 Forward genetic screen
EMS

F1 embryo
P0
+
+
m
heterozygous
for the mutation
+
 Forward genetic screen
EMS

P0

F1
 Forward genetic screen
50% have
mutant allele
•  Mendel’s 1st Law: The principle of (m)

equal segregation
gamete
production
–  two alleles of a gene pair segregate from one
50% have
another in the formation of gametes
wild-type allele
–  half of the gametes carry one allele, and the other (+)
half carry the other allele

F1
 Punnett Square

+ m + m or
+ m
+ + m m
homozygous
+ m wild-type heterozygous
mutant

1 : 2 : 1
+ + +

+ m
m m m

25% of F2 animals should exhibit a mutant phenotype

 1000
The progeny of how many F1 animals do you need to screen in order
to find a clone that carries a mutation in your gene of interest?
Looking for an F1
animal where 25%
of its progeny are
mutants of interest.

Look at 6-8 progeny of each F1 animal…

TOTAL: 6000-8000 F2 animals