INTRO TO DEVELOPMENTAL GENETICS ZEBRAFISH AS A MODEL SYSTEM Reading Skim Chapter 19 Slides from lecture will appear on the website Optional Supplementary reading Two review articles will also be posted one on the history of zebrafish as a model system and one on zebrafish genetic tricks and how these are used for mapping This reading is not required but available if you d like to supplement the lectures Problems Problem set and solutions posted on website Last lecture we talked about how mutagenesis strategies can be used to dissect biochemical pathways today we ll talk about how mutagenesis strategies can be used to study development DEVELOPMENTAL GENETICS Definition Study of how the fertilized egg of a multicellular organism becomes an adult Most developmental geneticists use simplier model organisms to uncover genes that are important in human development and disease Your book describes several model organisms Saccharomyces cerevisiae yeast Arabidopsis thaliana mouse ear cress a weed Caenorhabditis elegans a nematode worm Drosophila melanogaster fruit fly and Mus musculus the house mouse Today we ll highlight another model organism Danio rerio the zebrafish Zebrafish are freshwater vertebrates that share many of the features that make the other model organisms so amenable to genetic research In addition they have a few of their own little idiosyncracies the ability to make haploid and gynogenetic diploid embryos the optical clarity of the embryos that set them apart First let s review the features that make model organisms useful for genetic analyses 1 Easy to cultivate 2 Short generation time reproduce rapidly 3 Easy to house many individuals small size 4 Specific characteristics that make it particularly suitable for a given developmental process 5 Accumulated knowledge e g large numbers of mutations stock centers genomic tools including dense genetic maps genome sequencing projects WHAT FEATURES OF ZEBRAFISH MAKE IT USEFUL AS A MODEL GENETIC ORGANISM First zebrafish embryos develop outside the mother and are thus easily accessible for observation and experimentation In an embryo that is about 18 hours old you can see that it is already quite well developed with an eye ear and developing brain at the anterior and segmenting muscle and tail posteriorly The embryo is wrapped around a large yolk which nourishes the embryo until it can feed a few days later SLIDE 1 FIGURE OF 18 HOUR OLD ZEBRAFISH EMBRYO A second feature is that the zebrafish embryo is optically clear In a close up view of the trunk of a zebrafish we can see virtual transparency of zebrafish embryonic cells even more clearly In a high power view of the trunk region of a zebrafish embryo at about one day of development we can easily see structures that lie deep within the embryo For example we can see every forming notochord cell in the embryonic midline as well as each floor plate cell of the neural tube that lies directly above it At even higher power we can pick out the cell bodies of individually identifiable neurons in the spinal cord Thus the clarity of zebrafish embryos allows us to follow individual cells during development as well as to easily recognize developmental mutants in genetic screens simply by viewing them under a microscope SLIDE 2 HIGH MAG VIEW OF 24 HOUR ZEBRAFISH TRUNK A third feature is that zebrafish develop rapidly The blastula period lasts only 3 hours and gastrulation is completed in about 5 hours For those of you that haven t taken developmental biology the gastrula period of development is a time in which major cell rearrangements occur to transform the embryo into something that looks much more like an embryo to our eyes Mesodermal segmentation in which the mesoderm of the body is separated into a series of reiterated compartments occurs shortly afterwards so that by 24 hours segmentation is completed and many primary organ systems have formed By 72h the embryo has hatched from its eggshell and within the next 2 days will begin to actively hunt for food In a period of just four days the embryo has rapidly become a small version of the adult This rapid development obviously simplifies developmental and genetic studies For comparison a human zygote has only divided one time to make a two cell embryo in the first 24 hours after fertilization SLIDE 3 ZEBRAFISH STAGING SERIES A fourth advantage is that zebrafish adults reach sexual maturity quickly for a vertebrate having a generation time of about 10 weeks In addition a single pair of zebrafish can lay more than a hundred eggs at weekly intervals Zebrafish are very hardy fish and very easy to raise A fifth advantage is that there is an extensive genetic map of all 25 zebrafish chromosomes In addition there is a large effort to sequence zebrafish expressed sequence tags ESTs The EST database is a bank of mRNAs that are expressed during development Sequencing of the entire zebrafish genome is well underway SLIDE 4 EXAMPLES OF ZEBRAFISH MUTANTS Recently two large scale genetic screens for zebrafish developmental mutants have been performed These screens combined with several smaller scale screens have identified hundreds of genes involved in embryonic development Most of these mutants were identified strictly using morphological criteria To give you a flavor for the kinds of mutants that can be identified I showed a sampling of zebrafish developmental mutations at one day of development no tail mutants lack a tail and a notochord whereas cyclops and one eyed pinhead embryos are both cyclopic and deficient in head mesoderm From complementation tests we know that cyclops and one eyed pinhead are different genes A more subtle phenotype is revealed by a mutation in the no isthmus or noi gene At higher power one can see that noi mutant embryos lack the isthmus a structure which demarcates the division between the midbrain and hindbrain and later becomes the cerebellum no tail cyclops one eyed pinhead and no isthmus have now also been identified at the molecular level in large part with help of the zebrafish genetic map HOW DO WE SCREEN FOR ZEBRAFISH MUTANTS SLIDE 5 ZFISH TRADITIONAL VS HAPLOID SCREENING STRATEGIES Traditional approach In the parental P generation we mutagenize males their spermatogonia and mate to wild type females The F1 progeny are carriers of mutations induced in their father To generate families of fish that are carriers of a particular mutation F1 fish are crossed to wild type and the resulting F2 family is kept separate
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