Lab Experiment

Totipotency of the Unincubated Chick Blastoderm

(Adapted from "Laboratory Exercises in Developmental Biology" by Yolanda P. Cruz)

The domestic chicken (Gallus gallus domesticus) egg is a standard specimen in embryology and developmental biology. The embryo is relatively large and, more importantly, accessible during nearly all stages of development. Eggs are laid during later meroblastic cleavages, if the egg has been fertilized. A large body of basic information on chick development exists. The developmental stages have been standardized in a well known staging series by Hamburger and Hamilton (1951). Be sure to bring a copy of the chick embryo staging series with you to lab.

Totipotency refers to the ability of an embryonic cell to develop into a normal embryo in the absence of other embryonic cells normally associated with it. In many animals, only the zygote is totipotent. However, it has been known for some times that certain vertebrate embryos can be manipulated so that their constituent blastomeres, when properly isolated, exhibit developmental potency no less than that of the zygote. Such embryos are said to undergo regulative development.

The blastoderm is a disc of cells that lies on the surface of the yolky amniote egg. In the fertile chicken egg, the blastoderm typically has attained the blastula stage at the time of oviposition (egg laying). However, development is temporarily arrested until the egg is incubated at 99šF following oviposition. Because the embryonic body axes of the chick are determined after the blastula stage, the unincubated chick blastoderm provides an opportunity to test the totipotency of chick blastomeres at the blastula stage.

In this exercise, the blastoderm of a chicken egg will be bisected and incubated for 1 or 2 days to determine whether each half-blastoderm is capable of producing an embryonic axis, defined here as a primitive streak, and then by somites and neural tube. Further incubation may be possible if the manipulated embryos survive bacterial infection and mechanical damage. If at all possible, incubation beyond 3 days should be attempted because of the opportunity to observe the formation of somites, which in the chick proceeds at the rate of approximately one pair per hour. In addition, fusion of the neural folds proceeds at a similarly dramatic rate and is well worth the technical effort demanded by this exercise.

You will be asked to write a lab report about this experiment, so be sure to take good notes about your procedures and observations.

Procedure

1. Assemble the following materials: fertilized, unincubated eggs, straight dissecting needle for punching hole in air sac, egg holder; pair of dissecting forceps, bent wire microsurgical tool, slide coated with agar and neutral red dye, glass dish with chick Ringer solution, pipette and pipette bulb to wet agar slide, scalpel handle and blade for cutting agar chips, dissecting microscopes and fiber optics lights.

Common use Supplies: glass coverslips, jar of 70% ethyl alcohol, Kimwipes, melted paraffin, alcohol lamp, matches.

2. Open an unincubated, fertilized egg (using same procedure as last week). Stain the blastoderm with a chip of neutral red-impregnated agar. With your microsurgical tool, make a small puncture in the yolk on the side of the blastoderm. Insert the needle or point of the pin under the blastoderm until it presses against, but does not perforate, the vitelline envelope on the other side of the blastoderm. Carefully retract the needle so that its point cuts through the blastoderm and bisects it. Do not puncture or slit the vitelline envelope while doing this. Carefully withdraw the needle or pin from the yolk. Cover the hole in the eggshell with a glass coverslip and melted paraffin. Using a pencil, label your egg with a unique identifying name for each experiment. Return your egg to the incubator.

4. Repeat your experiment several time, or until the supply of eggs is expended. 5. Prepare at least one control embryo to which you make no attempt to divide the blastoderm.

6. Return after 24 hours to observe the embryo at this time using the vital dye neutral red to help you visualize your embryo, Return again at 48 hours. Depending on how aseptic the procedure was, the embryo should survive bacterial infection for at least 48 hours and beyond. During your observation, look for primitive streaks, somites, neural folds and neural tubes. Carefully record your observations (make notes and sketches of each embryo.)

Questions to Consider when your write the Discussion portion of your Lab Report

1. Explain how two axes would form from a bisected blastoderm. Consult Section 3.5 in our textbook as you think about this question. What does the ability to form two axes reveal about the totipotency of cells in the chick blastoderm?

2. How would you go about determining the maximum number of bisections that a chick blastoderm can sustain and still produce body axes?

3. What are the implications of blastoderm bisection for the formation of identical twins?

This page was posted 16 March 2003

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