ZEBRA, ZEBRA WHERE ARE YOUR STRIPES?

OBSERVING EMBRYOLOGICAL DEVELOPMENT

IN THE ZEBRAFISH

Barbara Tyler McGlone

Overview

How can one speak of the whole without considering the parts, or the very foundation upon which it is built? The complexity and uniqueness of man is not by chance, but has an organizational beginning. As Wolpert has emphasized, "it is not birth, marriage or death but gastrulation which is truly the most important time in your life." ¹

Who is the "new kid on the block"? Among the comparative studies and experiments of organisms such as the sea urchin, frog, nematode, mouse, Drosophila, Daphnia, squid and the chick, the zebrafish (Brachydanio rerio) with all his stripes has appeared on the scene, with much success! The zebrafish may prove to be the best comparison because the cleavage patterns are very easy to see, changes take place in a few hours and they are inexpensive. Generation time is 3-4 months. The females lay several hundred eggs that develop very rapidly. The study of the embryological development of the zebrafish will generate daily excitement as the students understand the uniqueness, similarities and the distinct differences of man and this organism.

Biological Concepts

• The embryological development of lower vertebrates

• Patterns of development (stages in development)

• Developmental hierarchy from three germ layers ( order of system development)

• Body plan of the embryo develops earl

Class Time

• Set up time: one class period (45 minutes)

• Laboratory write-up (20 minutes)

• Removal of eggs and observation (20 minutes)

• Daily observation and recording (15 minutes)

Background Information nd function can be mutated in the laboratory. The zebrafish has been used in many genetic experiments.

Materials

• zebrafish (24-30, equal number of males and females)

• 45L (10 gal) tank

• aquarium heater

• filter system

• embryo siphon

• 24 hour open tank aged water ( or chemically treated water)

• Paramecium

• Brine shirmp

• marbles

• embryological stages in development of the human and the zebrafish

• microscope (monocular, stereoscope and/or video microscope)

Teacher Preparation

1. Set up tank with aged ( or chemically treated) water at a level about 5cm from the

top.

(Alternative: Commercial sea salt - 60mg Instant Ocean/liter

chemically treated water.)

2. Set temperature to 28.5_C (Above 31_{_C and below 25_C the fish will}

_{not breed or develop normally.)}

_{3. Place 25 fish in the tank (equal number males -slender bodies with}

_{yellow bellies and females -plumper with}

_{silvery bellies).}

_{4. Replace 1/3 of the water each day.}

_{5. Siphon debris from the bottom if there is no filtering system.}

_{( If there is a filtering system , replace 1/2 the water weekly.)}

_{The siphon can be made from a plastic or glass tube (1cm - diameter,}

_{30-50cm - length) covered at one end with a piece of tygon tubing.}

6. Feed adults 1-2 times a day.( Brine shrimp )

(Clean the tank by siphoning after the second feeding.)

7. Prepare and /or display embryological development stages of the

human and the zebrafish.

Notes:

An automatic timer can be used to assure 14hrs of light and 10 hrs of day. Cover the tank with black plastic if the room is used at night. Provide a separate tank light insidet drape that is controlled by the automatic timer. Check water temperature and adjust if needed. To keep the adults from feeding on the newly spawned eggs cover the bottom of the tank with marbles. Periodically search the bottom of the tank for embryos and remove them. They should be placed in a seprate shallow container of the system's water. If you don't need the embryos let the fish rest by removing the marbles and feed them less food.

Otherwise, the fish will get fat and breed poorly.

Should you wish to raise the babies, keep them in the system's water about 3cm deep with 25-50 embryos per 100mL in a 250mL beaker (or 25 per 35mm Petri dish ) and change the water daily. The best temperature is 28.5_C. A temperature water bath with watch glass cover can also be used. The optimum hatching time is 3 days. Feed the larva live Paramecium the fourth day after fertilization. As the yolk decreases around the sixth day, increase the quantity. At the ninth day give the babies Paramecium and brine shrimp. Now you are ready to wean gradually to adult food. When they have gained in size, move to a larger container and take away the "baby food"(Paramecium). Feed twice a day and keep the fish per container low in number.

Resources

Heimer, Lennard, The Human Brain and the Spinal Cord, Functional Neuroanatomy

and Dissection Guide, 1995

Mathews, Willis W., Atlas of Descriptive Embryology, 4th edition, 1986.

Slack, J.M.W., From Egg to Embryo, 2nd edition, 1991.

Corley-Smith, G.E., Lim, C.J., & Branhorst, B.P., "The Zebrafish" Institute of

Molecular Biology and Biochemistry, Science Monitor, Vol 3(5) (www

"Fishnet")

Kimmell, Charles B., Ballard, William W., Kimmel, Seth R., Ullmann, Bonnie & Schilling, Thomas, "Stages of Embryonic Development of the Zebrafish",

Developmental Dynamics An Official Publication of the American Association of

Anatomists, Vol.203 No. 3 July 1995 (www "Fishnet")

Leff, David, "A Virus With a Backbone", Zebrafish, Mosaic, Vol 23, No.3,

Summer 1992

Walker, Charles and Streisinger, George, Eugene's The Zebrafish Book, A Guide

for Laboratory Use of the Zebrafish (www"Fishnet")

Cronkite, Donald, Professor of Biology, Hope College, PO Box 9000, E-mail

cronkite@hope.edu

Kanki, John, Department of Molecular Biology, Moffett Laboratory, Princeton

University, Princeton, NJ 08544, E-mail JKANKI@watson.princeton.edu

Reference

1

Slack, J.M.W., From Egg to Embryo, 2nd edition, copyright 1991, page 1

About The Author:

Barbara McGlone is a biology and anatomy and physiology teacher at Oscar Smith High School,

1994 Tiger Drive, Chesapeake, VA 23320.

****See Hardcopy for diagrams

Student Activity

NAME:______________________ DATE:__________PERIOD: _________

ZEBRA, ZEBRA WHERE ARE YOUR STRIPES?

OBSERVING EMBRYOLOGICAL DEVELOPMENT

IN THE ZEBRAFISH

Observation Lab

1. Prepare a formal laboratory report for Part I and Part II according to the following format:

Title:

Purpose:

Hypothesis:

Materials:

Procedure: (Number each step and place on separate lines)

Observations

A. Illustrate , color and label

B. Data Chart

C. Graph

Results/Analysis/Conclusion:

2. Using the siphon take the eggs out of the tank and put it in the Petri dish that contains a

small quanitiy of water that covers them.

3. Cover Petri dish.

4. Place the Petri dish on the platform of the stereoscope so that the eggs can be examined.

( A concave slide and a monocularscope can also be used.)

5. Observe and record observations of the eggs. Illustrate with labels and color. Use the

diagrams for identification of the stages in development that are visible.

6. Repeat numbers 2-5 for at least 4 days recording the stages observed.

7. Analyze results and write a conclusion, that includes the number of eggs removed and

the number of organisms developed. Describe the visibility and appearence of the organ systems in the zebrafish and make a comparasion with the early stages in development in humans.

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