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Using Zebrafish to Learn Statistical Analysis and Mendelian Genetics

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Using Zebrafish to Learn Statistical Analysis and Mendelian Genetics ZEBRAFISH Volume 8, Number 2, 2011 Original Articles ª Mary Ann Liebert, Inc. DOI: 10.1089/zeb.2010.0686 Using Zebrafish to Learn Statistical Analysis and Mendelian Genetics Samantha Lindemann,* Jon Senkler,* Elizabeth Auchter, and Jennifer O. Liang Abstract This project was developed to promote understanding of how mathematics and statistical analysis are used as tools in genetic research. It gives students the opportunity to carry out hypothesis-driven experiments in the classroom: students generate hypotheses about Mendelian and non-Mendelian inheritance patterns, gather raw data, and test their hypotheses using chi-square statistical analysis. In the first protocol, students are challenged to analyze inheritance patterns using GloFish, brightly colored, commercially available, transgenic zebrafish that express Green, Yellow, or Red Fluorescent Protein throughout their muscles. In the second protocol, students learn about genetic screens, microscopy, and developmental biology by analyzing the inheritance patterns of mutations that cause developmental defects. The difficulty of the experiments can be adapted for middle school to upper level undergraduate students. Since the GloFish experiments use only fish and materials that can be purchased from pet stores, they should be accessible to many schools. For each protocol, we provide detailed instructions, ideas for how the experiments fit into an undergraduate curriculum, raw data, and example analyses. Our plan is to have these protocols form the basis of a growing and adaptable educational tool available on the Zebrafish in the Classroom Web site. Introduction Many zebrafish mutant and transgenic strains are available commercially and from the Zebrafish International Resource ndergraduate laboratory courses provide valuable Center, and there is a wealth of information about how to Uopportunities for hands-on learning, the application of successfully raise and maintain them (zebrafish.org/zirc/ concepts learned in lectures and textbooks, and an introduc- home/guide.php).8–10 Our first protocol uses GloFish, a tion to the complexity of science and experimental design. brightly colored and fluorescent strain. GloFish are readily Mendelian genetics has long been taught through laboratory available in pet stores (www.glofish.com/), making it possi- experiments, with protocols for using Drosophila melanogaster ble to carry out challenging genetic experiments in schools in the classroom published as early as 1918.1 Here we present without access to a research zebrafish facility. two protocols that use the zebrafish model system to give Several excellent protocols already exist for using zebrafish students the opportunity to analyze Mendelian and non- to teach genetics (www.bioeyes.org/) (www.insciedout.org/) Mendelian inheritance patterns and rigorously test their hy- (www.glofish.com/classroom.asp).2–7 Our protocols build potheses through statistical analysis. The difficulty of these upon this earlier work by giving students background in how protocols can be easily adapted to students at different levels, genetic screens are used to find new mutants, bringing in the and the material requirements are low, making them acces- analysis of several phenotypes in a single cross, and including sible to a wide range of classrooms. inheritance patterns that do not fall into basic recessive and Zebrafish are being increasingly used for education, and in dominant patterns. Finally, these protocols add richness to the particular have many advantages for genetic experiments.2–7 study of genetics by challenging students to form their own They are easy to raise, and a single pair often produces 100 hypotheses, and then rigorously test these hypotheses through embryos or more when mated, making it possible for each the use of chi-square statistical analysis. Chi-square analysis student to have their own fish. Since adult fish live over 2 requires only the use of basic mathematic techniques, making years, the same fish can be used over many semesters, either it appropriate for middle school and high school students. directly when the adult fish express viable phenotypes or to Knowledge of mathematics is becoming even more important produce clutches containing mutant embryos for analysis. to biologists as they tackle increasingly complex problems and Department of Biology, University of Minnesota Duluth, Duluth, Minnesota. *These authors contributed equally to this work. 41 42 LINDEMANN ET AL. large data sets.11–13 This has led to a call for greater integration lected into deep (100Â20 mm) Petri dishes (Cat. #M090501; of mathematics into the undergraduate curriculum.13–15 Our Laboratory Products Sales, Rochester, NY), one dish for each protocols enable the introduction of mathematics into the clutch of eggs. To do this, the adult fish and the insert in the undergraduate curriculum during the freshman or sophomore mating tank were removed and the eggs allowed to settle. The year, providing a foundation for more complex mathematical majority of the water was poured out slowly so that no approaches in subsequent courses. eggs were lost. When about 20 mL of water was left, the re- The two protocols reported here were developed for a maining water and eggs were poured quickly into a Petri dish sophomore level undergraduate genetics laboratory course. (www.zfic.org/common%20techniques/mating.html). The This course met for one 4-hour laboratory session each week fertile eggs were sorted from infertile eggs and waste prod- of the semester, and all of the students (*60 students/ ucts using a dissecting microscope with transmitted light. semester, 10–20 students/laboratory section) either had taken Embryos were moved to the middle of the Petri dish using or were concurrently taking a genetics lecture course. How- the ‘‘embryo swirl’’ (www.zfic.org/common%20techniques/ ever, these experiments will coordinate with virtually every embryo%20swirl.html). The good eggs, which appeared genetics course, as they incorporate the concepts that form the translucent and had normal morphology, were sorted into basis of all genetics studies. In addition, these laboratories are one area of the Petri dish using an embryo loop (www student-driven and problem-based without being extremely .zfic.org/common%20techniques/Embryoloops.html), and labor intensive for the instructors. Thus, they will fit well into waste and infertile eggs removed. Healthy embryos were courses aiming to eliminate ‘‘cook-book’’ laboratories from moved to a new Petri dish containing clean aquatic system their curriculum. Further, the raw data we have included can water using a transfer pipet, and the Petri dish placed in an be used to introduce active learning and problem solving into incubator at 28.58C or in the aquatic fish facility until the fish lecture courses, which has been shown to have a positive were ready to be imaged or raised in a large tank. impact on the ability of students to retain knowledge and their long-term achievement.16 Our goal is to make this an evolving Scoring the phenotype of cyc and sqt mutants and growing collaborative protocol on our Zebrafish in the Between 2 and 3 dpf, developing embryos were counted Classroom Web site (www.zfic.org) that includes variations and scored by their eye phenotype using a dissecting micro- on how to use these ideas in the classrooms for students at scope, with normal embryos having two eyes and mutant diverse levels in their development as scientists. embryos having more closely spaced or cyclopic eyes. Materials and Methods Imaging All procedures have been approved by the University of Fish were photographed without anesthesia or with a short Minnesota IACUC Committee. A copy of the approved pro- incubation in 0.017% tricaine methanesulfonate (MS-222) tocol is available on the Zebrafish in the Classroom Web site dissolved in aquatic system water. Larvae were photo- (www.zfic.org/common%20techniques/IACUC-teaching-all graphed using a Leica S6 D stereo light microscope fitted with .pdf). In addition, students complete vertebrate animal safety a Nikon Digital Sight DS-SM camera. For all adult pictures training before starting the laboratories (Supplementary Ma- except the progeny in Figure 5 and all fish in Figure 14, the fish terial 1; Supplementary Data are available online at www were netted onto a flat surface with a small amount of water, .liebertonline.com/zeb). and then photographed using Panasonic DMZ-TZ3 digital camera. Images in Figure 5 were captured with a Canon EOS Rebel XS 18-55IS digital camera. For Figure 14, adult fish Fish stocks were netted onto the glass plate of an Olympus SZX12 Parental fish stocks included the wild-type (WT) strain stereomicroscope connected to a Cannon PowerShot A520 zebrafish Danio rerio (ZDR) (Aquatic Tropicals, Plant City, through one eye piece. Images were taken with white light FL), and strains carrying the following mutations and trans- illumination from above or with fluorescence microscopy. genes: cyclopsm294 (cyc)17; squintcz35 (sqt),18 mylz2:Yellow Fluorescent Protein (GloYFP),19 mylz2:Red Fluorescent Protein Results (GloRFP),19 golden (gol),20,21 and long fin (lof).22,23 Adult fish Laboratory 1: analysis of Mendelian inheritance were maintained using standard protocols.10 Fish to be raised patterns using GloFish were maintained for 8–9 days postfertilization (dpf) in Petri dishes at 28.58C, and then placed in a 10 L tank within a re- This laboratory is designed to be the opening laboratory of circulating, multi-rack aquarium system (Aquatic Habitats, the semester. Students analyze
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