THE PRODUCTION OF DOUBLE EMBRYOS 



A. THE EXPERIMENT: DOUBLE EMBRYOS BY INVERSION 



PURPOSE : To produce double monsters ty Inverting the egg In a gravitational field at the 

 two-cell stage, shifting the egg deutoplaam and thereby affecting subsequent gastrula- 

 tlon. 



MATERIALS : 



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

 In the 2-Gell stage. 



Technical : Standard equipment. 



METHOD : 



Precautions : 



a. Avoid excess handling if eggs and embryos. 



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



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



Controls : Eggs from the same source as the experl mentals, in the same stage of develop- 

 ment, adhered to the same kind of paper and in the same manner but without suffici- 

 ent 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 experiment. Cut some clean paper (both filter paper and white, smooth 

 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 determine 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 h 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 remain in- 

 verted, or were distorted by the Jelly-tension, It would be well to make a sketch 

 record In order to have a possible explanation of later developmental monstrosities. 



The original method of placing the eggs between glass plates (glass slides) and 



compressing them sufficiently to prevent rotation when the plates are inverted, cein 



be attempted. The objection to this method is simply that the pressure factor is 

 not uniform and should be taken into consideration. 



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