Zebrafish Developmental Genetics
Zebrafish characteristics
Largely rapidly developing transparent embryos
Large brood size and high re-mating frequency
Durable, easy to raise
Mutants stored as frozen sperm
External/in vitro fertilisation
Initially few markers, large genome, many
chromosomes
Generation – time: 3-4 months
Development
Genetic Screens
Zebrafish is the first vertebrate for which intensive mutagenesis has been
attempted – using other model organisms is difficult mammals, birds, xenopus) as
breeding takes time
Mutants are easily identified and parents can still be bred to keep mutation in the
heterozygotes (keeping the heterozygotes means that the mutant phenotype will
continue to be expressed and available for study)
Generation of Gynogenetic Haploid Embryos
Technique unique to fish eggs
1. Males are mutagenized and mated to wild-type females
2. Eggs from the F1 females are gently squeezed from females and
mixed with sperm treated with UV irradiation to destroy their
DNA
3. Irradiated sperm fertilise the eggs and activate their
development – these sperm do not give any genetic material to
the eggs
4. Eggs develop under the guidance of female genome only (gynogenetic haploid)
5. If the F1 female carried a mutant allele – would be seen in 50% of theses
embryos
6. Haploid embryos develop normally for about 24 hours and only live about 3 days
– so technique can be used only to screen for mutations of early development
(Cheng and Moore, 1997)
Mutagenesis and Screening
, Spontaneous mutations are rare (1 per 100 000 gametes) but
radiation/chemical mutagens can increase the mutation rate
(up to 1 per 300 gametes with ENU)
Developmental mutations are often lethal
In diploid organisms mutagenesis generates individuals which
are heterozygous for any new mutations
Heterozygous carriers survive but cannot be distinguished
from siblings
F2 Screen
G0: mutagenize males
F1: set up individual families
F2: within families – randomly mate with F2 siblings
F3: Identify families which produce F3 offspring
showing a mutant phenotype
1. Male parental fish treated with a chemical mutagen
(ENU) that will cause point mutations randomly in
germ cells
2. Each mutagenized male is mated to wild-type female
fish to generate F1 lines
3. Individual fish in the F1 generation carry mutations
inherited from his or her father – if mutation is
dominant, it will be expressed in the F1 generation
If mutations recessive, the F1 fish will not show a
mutant phenotype, since the wild-type allele will
mask the mutation
Each mutation is the product of an independent interaction of the mutagen with the
sperm DNA and is very likely to be unique.
4. The sibling F1 fish are mated to each other to produce the F2 generation – in this
generation some males and some females will have the mutated allele
5. F2 fish in each family are then mated to each other. When F2 parents contain the
same mutation as a recessive, there will be a 25% chance that their offspring will
show the mutant phenotype.
Since development occurs in the open, abnormal developmental stages are readily
observed (Driever et al., 1996)
Problems: time-consuming, tank and maintenance consuming, expensive, risky
Mutants recovered from F1 screens
cyclops: eyes close together, no floorplate
spade tail: tail defects
Zebrafish characteristics
Largely rapidly developing transparent embryos
Large brood size and high re-mating frequency
Durable, easy to raise
Mutants stored as frozen sperm
External/in vitro fertilisation
Initially few markers, large genome, many
chromosomes
Generation – time: 3-4 months
Development
Genetic Screens
Zebrafish is the first vertebrate for which intensive mutagenesis has been
attempted – using other model organisms is difficult mammals, birds, xenopus) as
breeding takes time
Mutants are easily identified and parents can still be bred to keep mutation in the
heterozygotes (keeping the heterozygotes means that the mutant phenotype will
continue to be expressed and available for study)
Generation of Gynogenetic Haploid Embryos
Technique unique to fish eggs
1. Males are mutagenized and mated to wild-type females
2. Eggs from the F1 females are gently squeezed from females and
mixed with sperm treated with UV irradiation to destroy their
DNA
3. Irradiated sperm fertilise the eggs and activate their
development – these sperm do not give any genetic material to
the eggs
4. Eggs develop under the guidance of female genome only (gynogenetic haploid)
5. If the F1 female carried a mutant allele – would be seen in 50% of theses
embryos
6. Haploid embryos develop normally for about 24 hours and only live about 3 days
– so technique can be used only to screen for mutations of early development
(Cheng and Moore, 1997)
Mutagenesis and Screening
, Spontaneous mutations are rare (1 per 100 000 gametes) but
radiation/chemical mutagens can increase the mutation rate
(up to 1 per 300 gametes with ENU)
Developmental mutations are often lethal
In diploid organisms mutagenesis generates individuals which
are heterozygous for any new mutations
Heterozygous carriers survive but cannot be distinguished
from siblings
F2 Screen
G0: mutagenize males
F1: set up individual families
F2: within families – randomly mate with F2 siblings
F3: Identify families which produce F3 offspring
showing a mutant phenotype
1. Male parental fish treated with a chemical mutagen
(ENU) that will cause point mutations randomly in
germ cells
2. Each mutagenized male is mated to wild-type female
fish to generate F1 lines
3. Individual fish in the F1 generation carry mutations
inherited from his or her father – if mutation is
dominant, it will be expressed in the F1 generation
If mutations recessive, the F1 fish will not show a
mutant phenotype, since the wild-type allele will
mask the mutation
Each mutation is the product of an independent interaction of the mutagen with the
sperm DNA and is very likely to be unique.
4. The sibling F1 fish are mated to each other to produce the F2 generation – in this
generation some males and some females will have the mutated allele
5. F2 fish in each family are then mated to each other. When F2 parents contain the
same mutation as a recessive, there will be a 25% chance that their offspring will
show the mutant phenotype.
Since development occurs in the open, abnormal developmental stages are readily
observed (Driever et al., 1996)
Problems: time-consuming, tank and maintenance consuming, expensive, risky
Mutants recovered from F1 screens
cyclops: eyes close together, no floorplate
spade tail: tail defects
