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Zebrafish Embryo Lab |
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Zebrafish Embryo Lab |
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The Zebrafish Embryo
Introduction
The zebrafish Danio rerio is a common aquarium fish from the tropics that we utilize for vertebrate developmental genetic study. What is the pattern of the early cleavages? How long are the cleavage cell cycles, and what fraction of the cell cycle is taken up by mitosis? How is morphology used to determine developmental time? What are the phenotypes of embryos bearing lethal patterning mutations? How is the vertebrate body plan represented in late embryos of this relatively simple vertebrate? These are some of the questions you can address and might be able to answer during this laboratory as you study the embryos and gain familiarity with what they look like as they progress through early development.
A nice feature of the embryo is that, along with its surrounding shell (chorion), it is highly transparent, such that by optical sectioning, you will be able to see much of the structure that is present. The accompanying drawings (Figure 1) show key stages that will help you in your exploration.
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Figure 1
A more complete set is available on ZFIN, the zebrafish research database located at http://zfin.org/zf_info/zfbook/stages/stages.html. Here are some landmark stages:
How would you characterize the division pattern of the early cleavages? How long is a typical cleavage cell cycle?
With a large bore pipette, transfer a few 2- or 4-cell embryos into a small petri dish, cover them entirely with water, and observe them with the dissecting microscope using transmitted, not reflected, illumination. Place the illuminating lamp rather close to the microscope to keep the fish embryos warm as they develop. An ideal temperature is about 28oC, several degrees above ambient room temperature. You will be able to gently roll the individual embryos about in the dish using a small tool such as a straight pin, but take care not to damage the chorion. Using this technique, you can observe the cleaving cells from different angles. It will help you figure out the cell arrangements and the way the cleavages occur. You will easily see that the early cleavages are incomplete, or "meroblastic", leaving the yolky region of the embryo uncleaved.
Can you come up with a simple rule that
predicts cleavage planes?
What is the nature of variability among individuals?
During late cleavage, after about the 5th or 6th division, the cells become so small and numerous that you will find it increasingly difficult to keep track of the cleavage pattern. Now is the time to move along to the next question.
What fraction of the cell cycle is taken up by mitosis?
A cell in interphase or very early prophase possesses a nucleus, but then the nucleus disappears and remains absent during the greater part of mitosis. Cell nuclei are readily visible when they are present in live blastomeres, so you can watch them come and go during the appropriate phases of the cell cycles. Do this and keep track of the time during at least a whole cell cycle.
You will need to use the compound microscope to visualize cell nuclei. Mount a late cleavage or blastula embryo, still in its shell, on a slide in a droplet of water beneath a coverslip bridge, made by using 3 coverslips in a stack to either side of the embryo that hold another coverslip above the embryo (7 in all) so that the embryo will not be squashed. Add enough water so that the embryo is completely covered and the preparation will not dry out quickly, but not so much water to make it into a sloppy mess. Use your desk light to warm the microscope stage and observe with an objective of your choosing (the 25x does nicely). You can increase the contrast by lowering the condenser from its normal position.
How is morphology used to determine developmental time?
Use the series of drawings to determine the ages of embryos we supply in the "mystery stage" beakers.
What are the phenotypes of embryos bearing lethal mutations that disrupt early development of specific body parts?
By the pharyngula period, the primary body organs have formed and many cell types have begun differentiation. For example, you will notice that pharyngula embryos can move, and movement requires both functional nerve cells and muscle cells. Hence, this time is an important one for screening mutagenized fish for patterning mutations. Prove this to your own satisfaction by identifying and examining in detail what has gone wrong with pharyngula embryos bearing cyclops and no tail mutations. What is wrong with the "mystery" mutant? You'll need to carefully compare mutants with their phenotypically wild type siblings, making your observations with the dissecting microscope, the tool with which the mutations were initailly discovered. We sketch key features of the wild type pharyngula in Figure 2.
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Figure 2
We'll supply beakers containing embryos of each strain that were obtained by intercrossing two fish heterozygous for the mutation in question.
If the mutations are recessive, what fraction of the embryos do you expect to look mutant? Do you suppose the mutations are, in fact, recessive?
Answer the same questions for no tail mutants.
We know that the no tail gene is the fish's homologue of the Brachyury gene of the mouse. Mouse Brachyury mutants have no tails and they have no notochords.
Anesthetize one or a few newly hatched larvae by using a pipette to transfer it to a dish containing a solution of 'MESAB' (MS-222 or Tricaine*). Anesthesia will occur in less than a minute. Transfer the animals to a slide in a droplet of the drug and mount it directly in the anesthetic with a 5-coverslip bridge arrangement (i.e. stack 2 on each side of the fishlet instead of 3 as you did earlier).
Caution: Do not splash the anesthetic around. It contains a nasty chemical, 3-aminobenzoic acid ethyl ester. Rather, be careful with it. Wear gloves if you like, but if you take some care, gloves will not be necessary.
Make your observations with both the dissecting and compound microscopes, transferring your preparation between the two as necessary to keep the morphology straight. During the course of your study, address the following questions:
* MESAB (MS-222 or Tricaine) - This is a very common fish anesthesia. It's chemical name is "MESAB, 3-amino-benzoic acid ethyl ester" and it can be ordered from Sigma (1-800-325-3010). One needs to be careful with MESAB. According to the Material Safety Data Sheet, it's potentially an irritant. We tell our students to exercise caution and to especially use gloves.
