1

Caenorhabditis elegans

Classical genetic analysis and cell signaling pathways

Basic biology and genetics

Developmental signaling

Epistasis analysis

Generation time 3 days

Brood size 200-300

Grow on bacterial lawn on petri dishesStore frozen @ -70C

Genome size 100Mb (sequenced 1998)

X0 males (<0.5%) XX hermaphrodites (self-fertilising)

F2 screen

m / +

+ / + OR m / +

+/+ X +/+ +/m X +/++/+ X m/+ +/m X m/+

G0 - mutagenise

F1 - individual mutants

F2 - generate male and female mutants

F3 - screen offspring

F2 screen

m / +

+/m X m/+

G0 - mutagenise

F1 - individual mutants

F2 - screen offspring

With self-fertilising hermaphrodites homozygous mutantscan be generated directly from F1 individuals

F2 - screen offspring

G0 - mutagenise hermaphrodites

F1 - self cross individual (mutant) hermaphrodites

2

Only 959 somatic cells in adult

Each cell has a unique identity

Body completely transparent

Development can be observed cell by cell in living worms

Each cell is the product of a unique lineage

Lineages invariant between individuals

All lineages have been mapped

AB = Neurons, Hypodermis, Pharynx, Body muscle

NeuronsHypodermisBody muscle

Gut

Germ cells

Body muscle

MS = Neurons, Hypodermis, Pharynx, Body muscle, Glands, Gonad

Structure and development of the vulva

8 primary vulval cells12 secondary vulval cells

Primary and secondary cells together form the vulval opening

6 tertiary hypodermal cells secrete the surrounding cuticle

Structure and development of the vulva

6 vulval precursor cells (VPCs) in the embryonic hypoderm

give rise to all 26 adult vulval cells

Structure and development of the vulva

One other cell (anchor cell) is required for vulva development

If the anchor cell OR all 6 VPCs are ablated no vulva forms

3

Saturation screening for vulva mutants

Wild type

No vulvaUsually recessive = loss of functionE.g. lin-3, let-60

Multiple vulvaeOften dominant = gain of functionE.g. let-23

Ordering mutations by epistasis analysis

Gene activities Phenotype

Wild type

Loss of function

Gain of function

Gain of function and loss of function mutationshave opposite effects on the phenotype

PHENOTYPE OF DOUBLE MUTANT?

Ordering mutations by epistasis analysis

PHENOTYPE OF DOUBLE MUTANT?

Gain of function in A and loss of function in B

Loss of function in A and gain of function in B

Ordering mutations by epistasis analysisWhen two mutations in a signaling pathway have opposite effects on the phenotype, the phenotype

of a double mutant will be that of the later acting gene

LET-23 IS DOWNSTREAM OF LIN-3

lin-3/lin-3 no vulvaLet-23D/+ multiple vulvaelet-60/let-60 no vulva

lin-3/lin-3 ; Let-23D/+ multiple vulvae

let-60/let-60 ; Let-23D/+ no vulvaLET-60 IS DOWNSTREAM OF LET-23

Ordering mutations by epistasis analysis

lin-3 is a secreted protein expressed in the anchor cell

let-23 is a transmembrane receptor expressed by VPCs

let-60 is an cytoplasmic protein expressed in VPCs

Limits to epistasis analysisImplicit assumptions

1. Genes act in a simple linear pathway

2. The pathway determines the final state of asingle end process, cell type or substance.

3. All mutations act as binary switches, jamming thepathway in either the OFF or the ON state.

4

Non-linear signaling pathways

αMSH (POMC)

MCR-1

non-agouti (a)

POMC/POMC yellow hair

a/a black hairAy/Ay yellow hair

MCR-1/MCR-1 yellow hairMCR-1D/+ black hair

Non-linear signaling pathways

POMC

MCR-1

a

POMC/POMC yellow hair

a/a black hairAy/Ay yellow hair

MCR-1/MCR-1 yellow hairMCR-1D/+ black hair

POMC/POMC ;MCR-1D/+

POMC/POMC ; a/a

MCR-1/MCR-1 ; a/a

black hair

yellow hair

yellow hair

SummaryC. elegans is a powerful model system for geneticsand developmental biology

Vulva development can be used to model cellsignaling genetics

Epistasis analysis is a classical approach to determining gene order

References

C. elegans development Molecular Cellular Biology 6th Edition pp 908-909

Vulva development Introduction to Genetic Analysis 9th edition pp 442

Epistasis analysis Molecular Cellular Biology 6th Edition pp 171-173