ABriefIntroduc/ontoZebrafish Genecs - Washington University Department of...
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Transcript of ABriefIntroduc/ontoZebrafish Genecs - Washington University Department of...
Outline
I. History of zebrafish as a model organism II. Gene/c screening in zebrafish
a) the first screens
b) “The Big Screen”
III. Other gene/c approaches in zebrafish
IV. Two personal vigneBes: using zebrafish gene/cs to study nervous system development.
I. History of zebrafish as a model organism
• George Streisinger – founding father
Streisinger’s history
• A member of the historic phage group at the dawn of the modern era of molecular gene/cs.
• Came of scien/fic age in the era where the focus was the gene and where great discoveries were made using muta/onal approaches in bacteria.
• Believed, like Brenner and Benzer, that the logic of increasingly complex systems could be deconstructed using muta/on-‐based gene/c analysis.
• Moved to the University of Oregon, Eugene, in 1960.
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Medaka
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Medaka
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Medaka Whitecloud Mountain Fish
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Medaka Whitecloud Mountain Fish
Why zebrafish?
• Streisinger wanted to carry out muta/onal analysis in vertebrates.
Medaka Whitecloud Mountain Fish Zebrafish
Why zebrafish?
• Breed very well in the laboratory – amenable to gene/c analyses
– breed year-‐round • External fer/liza/on
– gametes can be harvested separately
• Development is readily observable
Zebrafish development is readily observable (and fast)
Disadvantages of zebrafish
• Biggest obstacle: efficient recovery of mutant phenotypes in a diploid vertebrate.
• Need to iden/fy rare recessive muta/ons and propagate them in the (unaffected) heterozygous carrier. – C. elegans: single +/-‐ carriers can produce -‐/-‐ and +/-‐ siblings. – Drosphila had 50 years worth of gene/c tricks, like marked and
balancer chromosomes. • Lack of gene/c markers would make tracking affected
regions of the chromosome difficult. • Streisinger spent over a decade establishing zebrafish
(husbandry/embryology) and developing tools to quickly (one genera/on) recover recessive muta/ons from the germ line.
Streisinger et al. 1981 – the first cloned vertebrate
First efforts focused on the maternal germ line
• Streisinger’s landmark paper in 1981 described a highly efficient method for ac/va/ng the development of eggs without gene/c contribu/on from the sperm so that mutants could be recovered in one genera/on.
• Zebrafish can live ~3 days as haploid organisms, so this approach was useful to find muta/ons that affect embryonic development.
Haploid screens
gamma-‐ray More recently:
(UV cross-‐links DNA)
Haploid screen advantages
• Saves /me and money. • Mutants are recovered in one genera/on.
• No need to raise many F2 families.
• Useful for: – iden/fying changes in early development caused by muta/ons • muta/ons in mutagenized females • iden/fying muta/on-‐bearing heterozygous females
But haploid embryos are not perfect…
Produc/on of homozygous diploid embryos
• Haploid embryos can be made diploid by manipula/ng the embryo during early development.
• Streisinger first developed this technique because he wanted to use fish that were iden/cal to one another prior to mutagenesis in future gene/c screens.
Produc/on of homozygous diploid embryos “early pressure screens”
• Pressure 1.4 min amer fer/liza/on breaks down meio/c spindle, and egg keeps both sister chroma/ds.
• Even though all the genes in an EP diploid come from the same female, they are not homozygous at all loci because of cross-‐over in meiosis I. – useful for early mapping of genes
rela/ve to the centromere: the further a gene is from the centromere, the smaller the frac/on of mutant offspring. The closer the gene is to the centromere, the greater the chance of 50% mutant offspring
• 10-‐20% of EP-‐treated embryos develop abnormally because of physical damage to the eggs.
(Egg from a +/-‐ female)
(Eggs from a +/-‐ female completed meiosis I during ovula/on)
(Division and meiosis II triggered by sperm)
Produc/on of homozygous diploid embryos by heat shock
• Different from EP because meiosis II has occurred, and the haploid chromosomes are allowed to replicate – homozygous for every gene.
• Heat shock 15 min amer fer/liza/on inhibits mitosis.
• Eggs abort mitosis, and are now diploids.
• Half of the HS-‐treated embryos are mutant.
• >50% of embryos develop abnormally.
(Egg from a +/-‐ female)
(Eggs squeezed from a +/-‐ female completed meiosis I during ovula/on)
(Division and meiosis II triggered by sperm)
Emergence of a community
• Researchers in Eugene began to embrace the zebrafish
• In the mid-‐70s, Chuck Kimmel begins work on the zebrafish – neuroanatomy – describes more neurons in zebrafish than
had been recognized in any other vertebrate
– fate maps • Kimmel and Streisinger plan large scale
collabora/ve screens together to study paBerning and differen/a/on of the nervous system.
1927-‐1984
Early screens from Eugene: γ-‐ray-‐induced muta/ons
spadetail no tail cyclops
Early screens from Eugene: γ-‐ray-‐induced muta/ons
spadetail
Pisalls of γ-‐ray induced muta/ons
• Gene/c altera/ons that arise from ionizing radia/on vary – point muta/ons – large dele/ons* – transloca/ons*
* affect more than one gene
• Not ideal for satura/on screens: beBer to have a mutagen that induces lesions in single genes.
II. Zebrafish expand beyond Eugene: The “Big Screen”
Chris/ane Nüsslein-‐Volhard Wolfgang Driever
Max Planck Ins/tute Tübingen
MassachuseBs General Hospital Boston
Recapitulate the Drosophila screen for embryonic paBern mutants in a vertebrate.
Choice of mutagen: ENU
ENU: N-‐ethyl-‐N-‐nitrosourea
• Alkyla/ng agent: transfers its methyl group to nucleo/des.
• ENU was found to be more mutagenic in zebrafish than EMS.
• Pre-‐meio/c germ cells (spermatogonia) are mutagenized, not sperm.
• If mature sperm were mutagenized, muta/ons are not fixed, and progeny are mosaic.
Classic three-‐genera/on scheme Muta/ons induced in the parent genera/on are driven to homozygosity in the F3 genera/on.
P: Pre-‐meio/c spermatogonia are mutagenized
F1: non-‐mosaic heterozygotes each carrying one or more muta/ons.
F2: 50% of F2 animals are +/-‐ for the muta/on inherited from the F1 founder.
F3: F2 siblings are crossed, and homozygous mutant phenotype is seen in 25% of progeny.
“The Big Screen”
• The screen lasted from 1993 – 1995. • Between Tübingen and Boston, ~4000 embryonic lethal mutant phenotypes were recovered.
• Instead of all the data slowly trickling out, both groups published 37 papers in a single volume of Development.
Development Volume 123
A taste of the mutant phenotypes
• unique and essen/al func/ons • embryogenesis • epiboly • gastrula/on • dorsoventral paBerning • notochord forma/on • midline and body shape • somite forma/on and
paBerning • diges/ve organs • jaw and brachial arches • axon pathfinding • re/na development
• brain development • midbrain/hindbrain boundary
forma/on • forebrain development • neural survival • neural degenera/on • inner ear and lateral line • fin forma/on • cardiovascular system • hematopoiesis • craniofacial development • pigmenta/on • locomo/on
Going from mutant phenotype to muta/on
• Iden/fy candidate genes.
• Posi/onally clone the muta/on.
Candidate gene approach
• Assemble a collec/on of cloned genes that have some proper/es expected of the mutated locus.
• Test these genes as candidates: – Look at expression paBern – Look at mutant phenotype in another species
• Drawback: criteria for candidate gene selec/on are subjec/ve
Posi/onal cloning
• Unbiased approach applicable to any muta/on whose inheritance can be traced, even if nothing is known about the gene or biochemical pathways affected by the muta/on.
• Even though the genome is large, zebrafish are amenable to posi/onal cloning projects. – high fer/lity: allows analysis of several thousand meioses and fine mapping to a small interval
– external development: allows injec/on experiments to rapidly test candidate genes in an interval.
Posi/onal cloning – three steps
1. Iden/fy DNA segments (“markers”) that are near the mutant locus as judged by linkage analysis. -‐ simple sequence length polymorphisms (SSLPs); more
than 3500 primer pairs available commercially
2. Markers are correlated with genomic maps to iden/fy the physical region of the genome that contains the mutated gene (“the cri/cal region”).
3. Iden/fy the gene within the cri/cal region: -‐ sequence analysis -‐ morpholino phenocopy -‐ transgenic rescue of mutants with the wild type gene.
Tradi/onal posi/onal cloning in zebrafish
1. 1. Iden/fy DNA segments (“markers”) that are near the mutant locus as judged by linkage analysis. -‐ simple sequence length polymorphisms (SSLPs);
more than 3500 primer pairs available commercially -‐ also called CA-‐repeats, SSRs (simple sequence
repeats), microsatellites. -‐ length of the CA tract differs in different strains
8 different SSLP markers scored on pools of WT and mutant embryos:
Tradi/onal posi/onal cloning in zebrafish
2. Markers are correlated with genomic maps to iden/fy the physical region of the genome that contains the mutated gene (“the cri/cal region”).
Tradi/onal posi/onal cloning in zebrafish
2. Markers are correlated with genomic maps to iden/fy the physical region of the genome that contains the mutated gene (“the cri/cal region”).
Tradi/onal posi/onal cloning in zebrafish
2. Markers are correlated with genomic maps to iden/fy the physical region of the genome that contains the mutated gene (“the cri/cal region”).
marker 1 marker 2 marker 3
Tradi/onal posi/onal cloning in zebrafish
And many markers are scored in individuals to con/nue to narrow the region
2 recombinants
2 recombinants
0 recombinants
Tradi/onal posi/onal cloning in zebrafish
2. Markers are correlated with genomic maps to iden/fy the physical region of the genome that contains the mutated gene (“the cri/cal region”).
marker 1 2 rec.
marker 2 0 rec.
marker 3 2 rec.
But, if you join a zebrafish lab and get involved in a gene/c screen, you’ll probably never have to do this…
