INTRO TO DEVELOPMENTAL GENETICS: ZEBRAFISH AS A MODEL SYSTEMReading: Skim Chapter 19. Slides from lecture will appear on the website.Optional Supplementary reading: Two review articles will also be posted - one on the historyof zebrafish as a model system and one on zebrafish genetic "tricks" and how these are used formapping. This reading is not required, but available if you’d like to supplement the lectures.Problems: Problem set (and solutions) posted on websiteLast lecture we talked about how mutagenesis strategies can be used to dissect biochemicalpathways; today we’ll talk about how mutagenesis strategies can be used to study development.DEVELOPMENTAL GENETICSDefinition: Study of how the fertilized egg of a multicellular organism becomes an adult. Mostdevelopmental geneticists use simplier model organisms to uncover genes that are important inhuman development and disease.Your book describes several model organisms: Saccharomyces cerevisiae (yeast), Arabidopsisthaliana (“mouse ear cress”, a weed), Caenorhabditis elegans (a nematode worm), Drosophilamelanogaster (fruit fly), and Mus musculus (the house mouse). Today, we’ll highlight anothermodel organism, Danio rerio (the zebrafish). Zebrafish are freshwater vertebrates that sharemany of the features that make the other model organisms so amenable to genetic research. Inaddition, they have a few of their own little idiosyncracies (the ability to make haploid andgynogenetic 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 GENETICORGANISM?First, zebrafish embryos develop outside the mother and are thus easily accessible forobservation and experimentation. In an embryo that is about 18 hours old, you can see that it isalready quite well developed, with an eye, ear, and developing brain at the anterior, andsegmenting muscle and tail posteriorly. The embryo is wrapped around a large yolk, whichnourishes 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 trunkof a zebrafish, we can see virtual transparency of zebrafish embryonic cells even more clearly. Ina 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 everyforming notochord cell in the embryonic midline, as well as each floor plate cell of the neuraltube that lies directly above it. At even higher power, we can pick out the cell bodies ofindividually identifiable neurons in the spinal cord. Thus, the clarity of zebrafish embryosallows us to follow individual cells during development, as well as to easily recognizedevelopmental 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, andgastrulation is completed in about 5 hours. For those of you that haven’t taken developmentalbiology, the gastrula period of development is a time in which major cell rearrangements occurto 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 ofreiterated compartments, occurs shortly afterwards, so that by 24 hours, segmentation iscompleted and many primary organ systems have formed. By 72h, the embryo has hatched fromits eggshell, and within the next 2 days, will begin to actively hunt for food. In a period of justfour days, the embryo has rapidly become a small version of the adult. This rapid developmentobviously simplifies developmental and genetic studies. For comparison, a human zygote hasonly 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 morethan 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. Inaddition, there is a large effort to sequence zebrafish expressed sequence tags (ESTs). The ESTdatabase is a "bank" of mRNAs that are expressed during development. Sequencing of the entirezebrafish genome is well underway.****SLIDE #4: EXAMPLES OF ZEBRAFISH MUTANTS****Recently, two large scale genetic screens for zebrafish developmental mutants have beenperformed. These screens, combined with several smaller scale screens, have identifiedhundreds of genes involved in embryonic development. Most of these mutants were identifiedstrictly using morphological criteria. To give you a flavor for the kinds of mutants that can beidentified, I showed a sampling of zebrafish developmental mutations at one day ofdevelopment. no tail mutants lack a tail and a notochord, whereas cyclops and one-eyed pinheadembryos are both cyclopic and deficient in head mesoderm. From complementation tests, weknow that cyclops and one-eyed pinhead are different genes. A more subtle phenotype isrevealed by a mutation in the no isthmus, or noi, gene. At higher power, one can see that noimutant embryos lack the isthmus, a structure which demarcates the division between themidbrain 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 thezebrafish 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-typefemales. The F1 progeny are carriers of
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