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Developmental Biology Laboratory Observations of the Zebrafish Embryo

This lab will give you the opportunity to see cleavage divisions as they occur and to recognize significant stages of embryonic development of a simple vertebrate. The zebrafish, Danio rerio, is a common aquarium fish that breeds very easily. If fish are maintained in breeding condition, spawning occurs each day at dawn, and with fertilization external, all stages are easily accessible. The embryo and its outer shell are very clear optically, allowing for easy observation throughout its early development. The small size of the embryo allows you to section very deeply into the embryo with slight focus adjustments of the microscope to reveal much of the internal structural development. Development is also very rapid. The following table gives approximate times (after fertilization) of key developmental stages:

Part I. Observations of the Cleavage Period

During this period, the first 6 cleavages occur. The cells, or blastomeres divide synchronously at about 15 minute intervals. You can easily observe these early cleavages with the dissecting microscope, using transmitted light.

You will observe that the cleavages are partial; i.e. the region of the egg containing most of the yolk is not cleaved, only the yolk-free cytoplasm of the animal pole will show cleavage.  

  Figure 1

Usually, the first five cleavages are all vertical and occur at right angles to one another, as shown in the figure below.

Figure 2. View is from the animal pole with cleavage planes numbered. Irregular cleavages may occur; generally, they do not produce abnormal development

You will be given a sample of embryos in early cleavage. Take several for observation. Carefully observe your embryos using the small probe provided to orient the embryo so that you will be looking down on the animal pole. Determine the cleavage stage.

IMPORTANT NOTE: Try slight adjustments of your lighting to find the best viewing conditions. Sometimes slightly less lighting allows you to see more detail.

• Illustrate one of your embryos and indicate the cleavage stage you think it represents. Label the individual blastomeres with a small "b" and the cleavage planes by number (as in Fig. 2). Use the space provided on the observation sheet (pdf file).

• Carefully adjust your lighting to accentuate the nuclei. They are present during the first half of each cycle and their shapes change systematically. The nuclei are globular in very early interphase and become spherical by late interphase. As the cells enter mitosis, the nuclei are elliptical and then take on the shape of a rugby-style football shortly before they disappear during prophase. The mitotic chromosomes are more difficult to visualize.

Approximately 5 minutes after the nuclei disappear, you should see cleavage taking place.

IMPORTANT NOTE: It may be necessary to change to a monocular scope with greater magnification to see the nuclei.

To make this change, you will need to mount the embryo, still in its shell, on a slide in a generous drop of water beneath a coverslip bridge. You do this by stacking 3 coverslips on each side of the embryo and one above it (7 in all). This will prevent the embryo from being squashed. Observe with any objectives except the 100x oil immersion lens.

Go to Part 2
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