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casanova Mutants
The goal of this laboratory is to understanding and actively using the concepts of the scientific method. In addition, you will engage in practicing techniques of compound microscopy, including use of bright field microscopy as well as fluorescent microscopy. This experiment allows you to implement their knowledge of microscopy to real experimental data, providing you with a venue to employ your critical thinking and analytical skills. You will be asked to distinguish Casanova mutants from wild types using bright field microscopy. You will formulate your own hypothesis with regards to the effects of the mutation on the formation of the heart. Lastly, you will test this hypothesis while viewing the staged embryos under fluorescent microscopy and recording heart beats.
1. Tools
You will use several tools for the experiment today:
Compound fluorescent microscope. This is a standard workhorse microscope that can take high resolution images using bright field and fluorescent microscopy. Ideally, this microscope is attached to a digital camera that can take images and also short movies.
Embryo loops.These are for orienting and pushing the embryos around. These are made with fishing line, capillary tubes, and super glue. You should make several of these for yourself.
Glass Slides. Live embryos must be mounted on slides in methylcellulose before you can view them on the compound microscope.
Small Petri dishes You will view you embryos under the microscope while they are contained in the fish water filled Petri dishes.
Stereomicroscope for Bright Field. This is the most direct and probably most familiar method of light microscopy. In this method, the light comes directly through the sample. To adjust your stereomicroscope for bright field, you should turn on the light, and then adjust the mirror in the base until you see an equal, bright level of light throughout the field of view.2. Hypothesis
Based on your knowledge of zebrafish development and the organogenesis, devise your own hypothesis involving the effects of the cas mutant gene on development. Record your hypothesis in your notebook.***For instructor***
The instructor is urged to pose several questions to help students formulate hypotheses for this experiment. An example of a good question to form a hypothesis from would be: "Do the separate groups of heart precursors beat synchronously or asynchronously?"
For the learning purposes of this experiment, students should chose whether the heart beats synchronously or asynchronously and form a hypothesis statement corresponding to their choice.
3. Preparing the Embryos for Stereomicroscopy
The instructor has prepared embryos for this experiment. Wildtypes with the cas gene were crossed with each other to produce the embryos. Since the cas mutation is a recessive mutation, approximately one out of every four offspring from this type of mating will be a cas mutant. The other three will be phenotypically wildtypes, although two of them will carry the cas gene. For further exercise in understanding the mating, trying drawing a Punnet Square; the parents’ genotypes are cas/+ and cas/+. cas refers to the cas gene and the + refers to a normal gene. For more information on Punnet Squares, speak with your instructor.
You will a sample of embryos from your instructor. It will be your job to distinguish between the cas mutants and the wildtypes. Both the wild type and cas mutants have been crossed with the actin:GFP transgenic line, which will be useful during the fluorescence part of the exercise. For now, take the sample of embryos and place them into a Petri dish filled with fish water. NOTE: ALWAYS put your name and date on each Petri dish. The embryos require dechorionation to view key elements of development. Using a pair of fine forceps, carefully remove the chorion from the embryo. Does dechorionating make it easier of harder to see structures within the fish?
Why is it important to have wild type embryos crossed into the the actin:GFP transgenic line in addition to the Casanova mutant zebrafish embryos into the actin:GFP transgenic line? What purpose do the wild type embryos crossed into the the actin:GFP transgenic line serve?
4. Identifying cas Mutants using Bright Field Microscopy
Carefully, place the Petri dish containing one wild type and one cas mutant under a stereomicroscope. View the embryos using brightfield microscopy and look for the pericardial edema in the cas mutants. Having done the pre-lab for the experiment, you should be prepared to differentiate between the two embryos. You may use the embryo loops to move the embryos to different positions and angles.
What are the limitations of an observational approach to developmental biology? What are some of its advantages?
How does this change the brightness and contrast of the image you see? Try to adjust options to create different image fields. Are there structures that are easier to see in dark field versus bright field, and vice versa?
5. Preparing the Embryos for Compound Fluorescent Microscopy
Transfer methylcellulose onto a glass slide for the purpose of holding the embryos loosely in place (methylcellulose mounting protocol) Using a pipette, carefully transfer the embryo onto a glass slide and secure them in place. Turn on the compound fluorescent microscope and observe the embryo under blue light. At this point, you should be able to view the internal structures of the embryo which will appear green. At this time, adjust the embryo in a position that allows you to observe the internal organs of the embryo for a period of a minute.
What makes the internal embryo structures appear green? Did you experience any difficulties when positioning the embryo?
6. Testing the Hypothesis using Fluorescent Microscopy
It is recommended that you test your hypothesis by observing the bpm (beats per minute) of the cas mutant. To do this it would be handy to have some sort of time keeping device - a regular watch or cell phone would do just fine - in order to tell when 1 minute is up. Repeat this process for the wild type as well. Comparisons can be made between the casanova mutant heart beat and the wild type heart beat. Record all data in your notebook and use them to answer the discussion questions.
How long do you need to count each embryo to get accurate data?
How many embryos of each phenotype (wildtype versus casanova) will you need to count to be sure your results are accurate and consistent?
Discussion Questions
What are some differences you noted between the stereomicroscopes and compound microscopes?
Based on your observations, what major aspects of development does the cas mutant appear to be affecting?
Compare and contrast your images of the of wild type and cas mutant embryo. What are the major differences?
Did your data support your hypothesis?
Did you notice any patterns during your observation? (i.e. did the right heart beat faster than the left heart or vice versa?) If so, what can you infer about the nature of these patterns; how can you tell if any differences are significant?
Make sure to write down any other important conclusions that you may find.
What are some ways in which the experiment can be improved?