Download Insertional mutagenesis in zebrafish rapidly identifies genes

Insertional mutagenesis in zebrafish rapidly
identifies genes essential for early vertebrate
development
By Golling et. al
Presented by: Pam Lincoln
Background
•Previously, they used ethylnitrosourea (ENU) to make single
base pair mutations
• When they found a mutant of interest they tried to isolate
the gene
• Isolation of the mutation was challenging and often required
previous knowledge of the molecular pathways involved
Introduction
• Forward genetic screens are used to isolate genes necessary for
embryonic development
• Previous screens have shown about 800 genes can be mutated to
give relatively specific or localized defects during development
• Another 1600 genes can be mutated to give less specific
phenotypes
• In five and half years, from hundreds of mutants, the genes
underlying only about 50 mutants have been reported
• Using a new technique, mutated genes can be identified in as little
as two weeks
Methods
Mutagenesis:
• They used a Moloney murine
leukemia-based retroviral vector
as a mutagen for stable transfer
of exogenous genes and VSV-G
envelope protein to infect a wide
range of organisms
• They injected blastula stage
embryos with the retrovirus
• Founders were bred together and
F1 fish with multiple insertions
were used to generate an F2
generation
• Transgenic F2 were inbred and F3
were examined for mutations
Methods
Finding the Gene:
• A Southern was performed with DNA from embryos and adults
which were mated. They identified the band common to all
phenotypic embryos
• They performed inverse PCR with appropriate primers and isolate
the PCR product of the appropriate size
Methods (cont)
Genotyping Embryos:
• Embryos from heterozygous parents were sorted into
phenotypically wildtype and mutant groups
• They genotyped 24 embryos in each group using PCR
• Wildtype and Mutants yielded different PCR products
Alcian Blue staining
Results
Identification of retrovirus-induced mutations
Obtaining the Gene:
• When an insert is potentially linked to a mutant phenotype, the
DNA flanking the insert is cloned using inverse PCR. About 50% of
the time a candidate gene is found by homology search in the NCBI
database
• In additional cases small chromosomal walks are used to obtain
and sequence more DNA
• This yields a candidate gene 2/3 of the time
Results
Identification of retrovirus-induced mutations
Confirmation:
• To confirm that the correct gene was cloned they performed
linkage analysis
• Mutant embryos are homozygous, while wildtype embryos are
heterozygous or non-transgenic
• 24 mutants and 24 wildtype embryos were genotyped in most cases
• If there are no recombinants and no other insertions, the insert is
considered to be the cause of the mutation
• RT-PCR or in situ hybridization was used as further evidence that
the gene was disrupted by the appropriate insertion
Results
Classification of mutant phenotypes
Results
Classification of mutant phenotypes
• The first 75 mutants identified were listed
• Mutants were grouped by phenotypic defects based on examination
under a low powered microscope
• Classifications are considered preliminary
• Mutants were grouped in two classes, specific and general
• Specific mutations include those in which phenotypes are observed
in the brain, eyes, jaw, arches or cartilages, midline, ear, fins,
liver, gut, kidney, muscle, pigment, body shape, etc.
• General mutations include those with extensive cell death in the
central nervous system, a small head and eyes, show retardation
and those that show several defects
• Common and general defects were discarded in chemical
mutagenesis screens
Results
Classification of mutant phenotypes
• Hi954 - a mutant that results
from the disruption of a gene
encoding UDP-glucuronic acid
decarboxylase
• Cartilage cells do not stain,
but can be seen in crosssection
• Similar to an ENU-induced
mutant jekyll
Results
Classification of mutant phenotypes
• Hi2092 in the gene caudal
causes shortened trunk and
tail with no yolk extension
• Hi923 shows reduced
pigmentation in the body and
eyes due to a mutation in a
subunit of vacuolar ATP
synthase
Results
Genes required for early vertebrate development
Results
Genes required for early vertebrate development
• Two classes of mutants, broad and specific
• They suggest that genes required for protein synthesis, RNA
processing, DNA replication and chromatin assembly give rise to
non-specific mutations
• Genes required for transcription factors, receptors and ligands give
rise to mutants with specific developmental phenotypes
• 20% of mutants give rise to genes for which a biochemical function
cannot be predicted
Discussion
• ENU provided the first broad view of the types of embryonic and
early larval mutants that can be obtained and allowed estimates of
the number of genes required for development
• However, this method had a bias toward, receptors, ligands and
transcription factors
• The method described in this paper presents a less biased view of
the genes required for development
• Insertional mutagenesis makes the task of cloning the gene much
faster and easier but it initially requires substantially more work to
isolate the same number of mutations as ENU
Further Reading
Mullins, M. (2002). Building-blocks of embryogenesis. Nat Genet 31,
125-126.
Amsterdam, A. et al. A large-scale insertional mutagenesis screen in
zebrafish. Genes Dev. 13, 2713-2724 (1999).
http://www.genesdev.org/cgi/content/full/13/20/2713