280 LIMB FIELD OPERATIONS 



XENOPLASTIC LIMB TRANSPLANTATIONS 



These are transplantations between different genera and even more distantly related 

 (or separated) species. Using Amblystoma donors, such transplants may be attempted 

 to Bufo, Hyla, and Rana. In using Triturus donors, the student should first consult the 

 work of Twitty (1937), and others, which indicates that Triturus embryos produce a toxin 

 (from yolk) that paralyzes embryos of other genera. Depending upon the availability of 

 material, the following are suggested: 



a. Anuran belly ectoderm transplanted over the limb-field of a Urodele. 



b. Urodele limb-field (ecto- and mesoderm) transplanted to the post-gill region an 

 Anuran embryo in the tail-bud stage. 



c. Anuran belly ectoderm (stage #16-#17) transplanted for 24 hours over Urodele 

 limb-field mesoderm; then re-transplanted along with the urodele mesoderm, to 

 the flank region of Urodele embryos. This would clearly demonstrate the rela- 

 tion of foreign (xenoplastic) ectoderm and limb mesoderm in the formation of an 

 heterotopic limb. 



REGENERATION OF THE URODELE LIMB 



Newly developing limbs of Urodele larvae have remarkable powers of regeneration. 

 Amblystoma or Triturus larvae with limbs (stage #38 or older) should be anesthetized in 

 1/3, 000 MS 222 and the digits and limbs cut at various levels and angles, and be allowed 

 to regenerate. The following factors should be considered: 



a. Whether regenerative potencies can be eliminated by repeated extirpations. 



b. Whether the regenerated portion is structurally identical with that extirpated. 



c. Whether the level of the cut is the controlling factor in degree or perfection of 

 regeneration. 



d. Whether regeneration is achieved equally in limbs previously transplanted to 

 orthotopic or heterotopic positions. 



e. Whether limb regeneration is controlled by the associated girdle or nerve ele- 

 ments. 



In order to answer some of the above questions, it will be necessary to acquaint yourself 

 with the normal development and morphology of the limb and digits of the salamander. 

 Spalteholtz preparations of limbs and entire larvae should be made. Then, make the 

 following cuts on appropriate larvae and study the regeneration potencies. 



a. Cut off the right forelimb at the level of the wrist. 



b. Cut off the right forelimb halfway between the wrist and girdle, above the bend 

 of the elbow. 



c. Repeat "a" and "b" but make the cuts at the greatest possible angles. 



d. Make transverse cuts at the levels of "a" and "b" but extend the cut only halfway 

 through the limb (or wrist), leaving all parts attached. 



In each case, make a sketch of the limb and the extent of the cut. When regeneration is 

 complete, dissect out the parts to demonstrate the details of regeneration or clear the 

 larva by the Spalteholtz method. This method will show cartilage and bone development 

 but little information relative to nerve and muscle regeneration. 



OBSERVATIONS AND TABULATION OF DATA: 



Drawings, photographs, and preserved specimens constitute the record of the above ex- 

 periments on the limb field. It is most important that detailed records be kept, particu- 

 larly regarding the age and stage of the donor and of the host, the extent (areal and depth) 

 of transplants, the angles of cuts, etc. Confirmatory histological analysis is excellent 

 but is not always necessary. It is more important that the student carries out thoroughly 

 one of the above procedures rather than to attempt parts of the entire exercise. 



