Caenorhabditis elegans – classical genetics and cell signalling
Sydney Brenner (1963) envisioned a model system which could allow the study of
post-molecular biology, development, neurobiology – something that can’t be done
with single cells
John Sulston – successfully campaigned or full genome sequence (1998) – allowed
further advances in studies of cell death, cell signalling, ageing, RNAi technology
Characteristics (David, 1997, Horvitz, 1997)
~1mm in length, free-living, non-parasitic soil nematode
Transparent for ease of manipulation and observation
Feeds on bacteria (eg. E.coli)
Easily and cheaply housed, cultivated in large numbers (10 000 worms/petri dish)
- Grow on bacterial lawn on petri dishes
- Can store frozen at -70oC
Genome size small = 100Mb – transcriptomes and proteomes also available
Invariant cell lineage, wild-type individuals contain constant 959 cells
- Position of cells is constant as is the cell number (are there mutants where cell
lineage becomes stochastic?)
3 day life cycle (from egg to sexually mature adult, but can live for longer – 14
weeks)
- Hard exoskeleton – go through several larval moults
Simple nervous system
5 pairs of autosomes, 1 pair of sex chromosomes – 2 sexes, hermaphrodites and
males (sexual determination similar to Drosophila, ratio of sex chromosomes to
autosomes determines its sex)
- If 6th chromosome pair is XX C. elegans is a hermaphrodite, XO is male
- Hermaphrodites can mate with males but cannot fertilise each other –
hermaphrodites the most common sex (males <0.5%) hermaphrodites mate
with males = 50% of progeny will males and 50% hermaphrodites
- Males produced when there is an error in meiosis
- In the lab – self-fertilisation can produce about 300-350 offspring and more
when mating with males traits make it easy to produce numerous genotypes
and phenotypes for genetic research
Techniques possible: systematic chemical mutagenesis, serial electron micrographs
(full description of anatomy), lineage tracing
Standard F2 screen is much more straightforward – skip a step as hermaphrodites can
self-fertilise
, Cut out a whole generation – in a normal
situation the F2 mutants are
heterozygous and another generation
must be produced before screening for
phenotypes
Due to self-fertilisation – skips whole
generation easy for saturation
mutagenesis
Invariant cell lineage of 959 somatic cells in adult…
Each cell has a unique identity, completely
transparent – development can be observed
cell by cell in living worms
Each cell is the product of a unique lineage
- Lineage invariant between individuals
- All lineages have been mapped
- Fates of cells become more and more specified
throughout development
Structure and development of the vulva
8 primary vulval cells and 12 secondary
vulval cells, 6 – primary and secondary
cells together form vulval opening
6 tertiary hypdermal cells secrete the surrounding cuticle
In the embryo – 6 vulval precursor cells in the
embryonic hypoderm which gives rise to all 26
adult vulval cells
An anchor cell is also required for vulva
development – if the anchor cell or all 6
equipotential VPCs are ablated, no vulva forms
- Use laser gun to ablate specific cells
- If some VPCs are left behind – they are able to
change its programme and generate all 26 adult
cells = PVCs are an equivalence group
Saturation screening for vulva mutants
Look for mutants that where the initial signal has been disrupted
, No vulva (Vul phenotype) mutants with defective vulva induction, usually recessive
(eg. mutations that decrease activity of the EGf-receptor let-23… lin-3, let-60)
Other mutants – protruding vulvae/egg-laying defective, abnormal eversion of vulva
Multivulva (Muv) phenotype – often dominant = gain of function (mutations that
activate Let-23) – multi-vulva hermaphrodites typically have a single functional vulva
and additional ventral protrusions, each a pseudovulva formed from vulval tissue
Vulvaless hermaphrodites have fertile eggs
Fact that multiple loci gives the same phenotype suggests they are all involved the
development of the vulve – possible questions: in what order do these genes act? Is
there a signalling or regulatory pathway involved?
Epistasis Analysis
Epistasis: masking the effect of one gene by
another gene
Analysis can be used to determine a functional
order of action of 2 genes – regardless of the
directness of interactions
- Most informative when genes analysed control
a common process – important to determine
relationships between mutations of interest
before trying to construct formal genetic
pathway
Two different kinds of pathways exist
- Switch regulation pathway: genes or gene
products that can be turned on/off, different
states of the genes determine outcome of the
pathway – genes involved in this type of pathway
will have 2 distinct and opposite phenotypes, will
also have the ability to bypass the requirement for upstream genes
- Substrate dependent pathway: involves a substrate, where an obligate series of
sequential steps are required to generate the final outcome – examples include
metabolic pathways and bacteriophage morphogenesis
Mutations in genes involved in the substrate dependent pathway will have
phenotypes that suggest a progression of events
Back to vulval development… (lin = lineage abnormal, let = lethal)
When 2 mutations in a signalling pathway have opposite effects on the phenotype,
the phenotype of a double mutant will be that of the alter acting gene
lin-3/lin3 no vulva lin-3/lin-3 & Let23D/+ multiple vulvae
, let-23D/ + multiple vulvae let-60/let-60 & Let23D/+ no vulva
let-60/let-60 no vulva
Conclusions: Let23 is downstream of lin3, let-
60 is downstream of let23 =
Lin3 Let23 let-60
Lin-3 is a secreted protein expressed in the
anchor cell
Let-23 is a transmembrane receptor
expressed by VPCs
Let-60 is a cytoplasmic protein expressed in
VPCs
Genetic redundancy can complicate analyses
Limits to epistasis analysis
Merely provides a working model for more
phenotypic studies or molecular analysis – not
necessarily the “answer”
1. Genes act in a simple linear pathway
- Some pathways are branched or contain multiple inputs
- Some pathways use tissue specific regulators not necessarily
used in other developmental processes that use a subset of
these same genes
- Some pathways may also have more than one
input
Sex-determination pathway in C. elegans
- High X chromosome to autosome ration (X:A) negatively regulates fem-1, fem-2,
fem-3 genes, which negatively regulate tra-1 gene
- Tra-1 functions to activate the developmental program for the female soma while
repressing the developmental program for the male soma
- However, role of fem-1, fem-2, fem-3 gene products don’t just regulate tra-1 –
genes also responsible for promoting male germline development and negatively
regulating female germline development
- Branch point at fem-1,2,3
2. The pathway determines the final state of a
single end process, cell type or substance
Sydney Brenner (1963) envisioned a model system which could allow the study of
post-molecular biology, development, neurobiology – something that can’t be done
with single cells
John Sulston – successfully campaigned or full genome sequence (1998) – allowed
further advances in studies of cell death, cell signalling, ageing, RNAi technology
Characteristics (David, 1997, Horvitz, 1997)
~1mm in length, free-living, non-parasitic soil nematode
Transparent for ease of manipulation and observation
Feeds on bacteria (eg. E.coli)
Easily and cheaply housed, cultivated in large numbers (10 000 worms/petri dish)
- Grow on bacterial lawn on petri dishes
- Can store frozen at -70oC
Genome size small = 100Mb – transcriptomes and proteomes also available
Invariant cell lineage, wild-type individuals contain constant 959 cells
- Position of cells is constant as is the cell number (are there mutants where cell
lineage becomes stochastic?)
3 day life cycle (from egg to sexually mature adult, but can live for longer – 14
weeks)
- Hard exoskeleton – go through several larval moults
Simple nervous system
5 pairs of autosomes, 1 pair of sex chromosomes – 2 sexes, hermaphrodites and
males (sexual determination similar to Drosophila, ratio of sex chromosomes to
autosomes determines its sex)
- If 6th chromosome pair is XX C. elegans is a hermaphrodite, XO is male
- Hermaphrodites can mate with males but cannot fertilise each other –
hermaphrodites the most common sex (males <0.5%) hermaphrodites mate
with males = 50% of progeny will males and 50% hermaphrodites
- Males produced when there is an error in meiosis
- In the lab – self-fertilisation can produce about 300-350 offspring and more
when mating with males traits make it easy to produce numerous genotypes
and phenotypes for genetic research
Techniques possible: systematic chemical mutagenesis, serial electron micrographs
(full description of anatomy), lineage tracing
Standard F2 screen is much more straightforward – skip a step as hermaphrodites can
self-fertilise
, Cut out a whole generation – in a normal
situation the F2 mutants are
heterozygous and another generation
must be produced before screening for
phenotypes
Due to self-fertilisation – skips whole
generation easy for saturation
mutagenesis
Invariant cell lineage of 959 somatic cells in adult…
Each cell has a unique identity, completely
transparent – development can be observed
cell by cell in living worms
Each cell is the product of a unique lineage
- Lineage invariant between individuals
- All lineages have been mapped
- Fates of cells become more and more specified
throughout development
Structure and development of the vulva
8 primary vulval cells and 12 secondary
vulval cells, 6 – primary and secondary
cells together form vulval opening
6 tertiary hypdermal cells secrete the surrounding cuticle
In the embryo – 6 vulval precursor cells in the
embryonic hypoderm which gives rise to all 26
adult vulval cells
An anchor cell is also required for vulva
development – if the anchor cell or all 6
equipotential VPCs are ablated, no vulva forms
- Use laser gun to ablate specific cells
- If some VPCs are left behind – they are able to
change its programme and generate all 26 adult
cells = PVCs are an equivalence group
Saturation screening for vulva mutants
Look for mutants that where the initial signal has been disrupted
, No vulva (Vul phenotype) mutants with defective vulva induction, usually recessive
(eg. mutations that decrease activity of the EGf-receptor let-23… lin-3, let-60)
Other mutants – protruding vulvae/egg-laying defective, abnormal eversion of vulva
Multivulva (Muv) phenotype – often dominant = gain of function (mutations that
activate Let-23) – multi-vulva hermaphrodites typically have a single functional vulva
and additional ventral protrusions, each a pseudovulva formed from vulval tissue
Vulvaless hermaphrodites have fertile eggs
Fact that multiple loci gives the same phenotype suggests they are all involved the
development of the vulve – possible questions: in what order do these genes act? Is
there a signalling or regulatory pathway involved?
Epistasis Analysis
Epistasis: masking the effect of one gene by
another gene
Analysis can be used to determine a functional
order of action of 2 genes – regardless of the
directness of interactions
- Most informative when genes analysed control
a common process – important to determine
relationships between mutations of interest
before trying to construct formal genetic
pathway
Two different kinds of pathways exist
- Switch regulation pathway: genes or gene
products that can be turned on/off, different
states of the genes determine outcome of the
pathway – genes involved in this type of pathway
will have 2 distinct and opposite phenotypes, will
also have the ability to bypass the requirement for upstream genes
- Substrate dependent pathway: involves a substrate, where an obligate series of
sequential steps are required to generate the final outcome – examples include
metabolic pathways and bacteriophage morphogenesis
Mutations in genes involved in the substrate dependent pathway will have
phenotypes that suggest a progression of events
Back to vulval development… (lin = lineage abnormal, let = lethal)
When 2 mutations in a signalling pathway have opposite effects on the phenotype,
the phenotype of a double mutant will be that of the alter acting gene
lin-3/lin3 no vulva lin-3/lin-3 & Let23D/+ multiple vulvae
, let-23D/ + multiple vulvae let-60/let-60 & Let23D/+ no vulva
let-60/let-60 no vulva
Conclusions: Let23 is downstream of lin3, let-
60 is downstream of let23 =
Lin3 Let23 let-60
Lin-3 is a secreted protein expressed in the
anchor cell
Let-23 is a transmembrane receptor
expressed by VPCs
Let-60 is a cytoplasmic protein expressed in
VPCs
Genetic redundancy can complicate analyses
Limits to epistasis analysis
Merely provides a working model for more
phenotypic studies or molecular analysis – not
necessarily the “answer”
1. Genes act in a simple linear pathway
- Some pathways are branched or contain multiple inputs
- Some pathways use tissue specific regulators not necessarily
used in other developmental processes that use a subset of
these same genes
- Some pathways may also have more than one
input
Sex-determination pathway in C. elegans
- High X chromosome to autosome ration (X:A) negatively regulates fem-1, fem-2,
fem-3 genes, which negatively regulate tra-1 gene
- Tra-1 functions to activate the developmental program for the female soma while
repressing the developmental program for the male soma
- However, role of fem-1, fem-2, fem-3 gene products don’t just regulate tra-1 –
genes also responsible for promoting male germline development and negatively
regulating female germline development
- Branch point at fem-1,2,3
2. The pathway determines the final state of a
single end process, cell type or substance
