THE ORIGIN OF AMPHIBIAN PIGMENT 



PURPOSE : To experlmentaHy test the thesis, by excision, explanation, homoplastic and 

 heteroplastic transplantation, that the amphibian pigment is derived from the neural 

 crests. 



MATEBIALS : 



Biological : Urodele larvae stages #13 to #3!+ 

 Anuran larvae stages #14 to #l8 



Technical : Standard operating equipment. 



Glass tubing measuring 0.25 to 0.14-5 mm. in diameter. 

 Petri dishes, depression slides, cover slips. 



METHOD: 



Precautions : 



1. Sterile precautions will Insure greater success, although amphibian tissues are 

 veiy hardy. This Is particularly true of the isolation cultures. The media 

 should be boiled or autoclaved; the Instruments sterilized either by boiling or 

 in alcohol; and the denuded embryos should be put through several changes of 

 sterile medium to remove surface bacteria. 



Controls : 



1. For the excision experiments the control consists of excision of any region other 

 than the neural crest. 



2. For isolation experiments of neural crest anlage, the control likewise consists 

 of the isolation of any region other than the neural crest, and the tissue may 

 be taien from the same donor. 



3. For transplantation experiments the control consists of the transplantation of 

 regions other than the neural crest to the same locality in the host as the 

 experimental transplant. 



Procedure : The following descriptions will apply specifically to Amblystoma but may 

 be followed with comparable stages of Anuraji material. 



EXCISION OF THE NEURAL CREST 



The neural crest may be easily excised at the neural fold stage, before the folds 

 have come together, at stage #15 for the Urodele embryo (or stage #14- for Anura). 



1. Eemove the membranes from Urodele lai-vae, stage #l'+-#15 In Operating Mediiun. 



2. Place the embryo in a shallow depression in Permoplast or paraffin and, using 

 a double-edged lancet or needles, excise the neural fold on the right side as 

 indicated in the accompanying diagrams. Include the dorsal ectoderm. 



3. Leave the embryo in Operating Medium until the wound closes over, then transfer 

 it to Growing Medium at about 15°-l8°C. and allow it to develop. A single speci- 

 men in a #2 Stender or finger bowl is best. 



This operation will have the more graphic results in the more highly pigmented 

 Amblystoma tlgrinum or T. pyrrhogaster. 



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