35. THE ORIGIN OF AMPHIBIAN PIGMENT 



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

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

 crests. 



MATERIALS: 



Biological: Urodele larvae stages #13 to #34 

 Anuran larvae stages #14 to #18 



Technical : Standard operating equipment. 



Glass tubing measuring 0. 25 to 0. 45 mm. in diameter. 

 Petri dishes, depression slides, cover slips. 



METHOD: 



Precautions : 



1. Sterile precautions will insure greater success, although amphibian tissues 

 are very 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 con- 

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

 may be taken 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 ex- 

 perimental transplant. 



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

 be followed with comparable stages of Anuran 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. Remove the membranes from Urodele larvae, stage #14-#15 as follows: Fresh clus- 

 ters of Amblystoma eggs may be dipped briefly into 1% KOH, rinsed thoroughly, and 

 quickly placed in isotonic medium (urodele medium, pond or spring water). With for- 

 ceps or wide-mouthed pipette remove the individually capsulated embryos from their 

 more inclusive but soft jelly masses. Then with pipette transfer these encapsulated 

 embryos to 0. 5% sodium -sulfadiazine (in pond or spring water or urodele medium) 

 and leave them for 30 minutes. Finally, transfer the embryos to finger bowls with a 

 ratio of 4 cc of medium per embryo (25 per finger bowl of 100 cc medium). Avoid 

 unnecessary crowding. The embryos are then ready for the final decapsulation with 

 sharp, watchmaker's forceps, in anticipation of the surgical procedures. 



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. 



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