A Brief Introduc/on to Zebrafish Gene/cs. 2 February 2012 hbp://monklab.wustl.edu

Transcription

1 A Brief Introduc/on to Zebrafish Gene/cs 2 February 2012 monkk@wustl.edu hbp://monklab.wustl.edu

2 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.

3 I. History of zebrafish as a model organism George Streisinger founding father

4 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.

5 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates.

6 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates. Medaka

7 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates. Medaka

8 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates. Medaka Whitecloud Mountain Fish

9 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates. Medaka Whitecloud Mountain Fish

10 Why zebrafish? Streisinger wanted to carry out muta/onal analysis in vertebrates. Medaka Whitecloud Mountain Fish Zebrafish

11 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

12 Zebrafish development is readily observable (and fast)

13 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.

14 Streisinger et al the first cloned vertebrate

15 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.

16 Haploid screens gamma- ray More recently: (UV cross- links DNA)

17 Haploid screen advantages Saves /me and money. Mutants are recovered in one genera/on. No need to raise many F 2 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

18 But haploid embryos are not perfect

19 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.

20 Produc/on of homozygous diploid embryos early pressure screens (Egg from a +/- female) (Eggs from a +/- female completed meiosis I during ovula/on) (Division and meiosis II triggered by sperm) 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.

21 (Egg from a +/- female) (Eggs squeezed 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.

22 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.

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24 Early screens from Eugene: γ- ray- induced muta/ons cyclops no tail spadetail

25 Early screens from Eugene: γ- ray- induced muta/ons spadetail

26 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.

27 II. Zebrafish expand beyond Eugene: The Big Screen Chris/ane Nüsslein- Volhard Max Planck Ins/tute Tübingen Wolfgang Driever MassachuseBs General Hospital Boston Recapitulate the Drosophila screen for embryonic pabern mutants in a vertebrate.

28 Choice of mutagen: ENU

29 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.

30 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.

31 The Big Screen The screen lasted from 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.

32 Development Volume 123

33 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

34 Going from mutant phenotype to muta/on Iden/fy candidate genes. Posi/onally clone the muta/on.

35 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

36 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.

37 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.

38 Tradi/onal posi/onal cloning in zebrafish 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

39 8 different SSLP markers scored on pools of WT and mutant embryos:

40 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 ).

41 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 ).

42 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

43 Tradi/onal posi/onal cloning in zebrafish And many markers are scored in individuals to con/nue to narrow the region 2 recombinants 0 recombinants 2 recombinants

44 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.

45 But, if you join a zebrafish lab and get involved in a gene/c screen, you ll probably never have to do this