23. THE PRODUCTION OF DOUBLE EMBRYOS 



A. DOUBLE EMBRYOS BY INVERSION 



PURPOSE: To produce double monsters by inverting the egg in a gravitational field at 



the two-cell stage, shifting the egg deutoplasm and thereby affecting subsequent gas- 

 trulation. 



MATERIALS: 



Biological: Ovulating Rana pipiens and adult males of the same species; Urodele 

 eggs in the 2-cell stage. 



Technical: Standard equipment. 



METHOD: 



Precautions : 



1. Avoid excess handling of eggs and embryos. 



2. Avoid desiccation of eggs, crowding, and heat. 



3. Practice adhering eggs to filter and glazed paper before experimenting. 



Controls: Eggs from the same source as the experimentals, in the same stage of de- 

 velopment, adhered to the same kind of paper and in the same manner but with- 

 out sufficient tension to prevent rotation of the egg within its membranes. These 

 controls may be inverted along with the experimentals, but they must be able to 

 rotate within their membranes. 



Experimental Procedure: 



Ovulate a female Rana pipiens and fertilize the eggs about 2 hours before the 

 time of the experinient. Cut some clean paper {both filter paper and white, 

 smooth glazed paper) into 1 inch squares and practice adhering eggs with their 

 jelly capsules to this paper. Place one egg only on each piece of paper. The 

 egg is transferred to the square of paper with a minimum of water. Then, using 

 a scalpel, spread the egg jelly down onto the paper in all directions in such a 

 manner that the drying jelly will hold the egg firmly to the paper. Allow the 

 jelly to dry slightly, in air. Test by inverting the paper and the attached egg 

 over a finger bowl of culture medium for 2 minutes and then re-examine to de- 

 termine whether the egg has rotated or has been held firmly in the inverted 

 position. Remember that there must be some tension to hold the egg sufficiently 

 to prevent rotation. 



Prepare several finger bowls of culture medium and quickly adhere 2-cell stages 

 to the single pieces of paper in the manner described. In all cases orient the 

 egg so that the animal pole is uppermost. After making certain that there is 

 sufficient tension to prevent rotation, invert the paper, with adherent egg, in 

 the finger bowl of culture medium and leave it undisturbed through at least the 

 two subsequent cleavage as determined by parallel-developing control eggs. If 

 eggs are mounted separately they may be examined briefly after the completion 

 of the second cleavage, and those which do not remain inverted should be so 

 marked or discarded. The pieces of paper float and the eggs are adequately 

 submerged in the medium. 



After the 8-cell stages has been achieved by the controls (about 4 hours after 

 insemination) carefully remove all of the experimental eggs from their paper 

 squares and place them separately in #2 Stenders. If particular eggs did not 



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