TRANSPLANTATIONS 



255 



h. Prepare the host by cutting a square hole with a glass needle (or a lancet) 

 about 5 somites In diameter at the level of the pronephric bulge but just beneath 

 the somites #8 to #10. The excavation nnist be deep enough to include some under- 

 lying mesoderm. This may be done with a hair loop. 



5. Prepare the donor forelimb anlage by excising the limb area, including the 

 underlying mesoderm. This can be done by passing a glass needle from ventral to 

 dorsal beneath the body ectoderm Just posterior to the gill buds, and dorsal to (and 

 including) the pronephric bulge. By bringing the needle upwards, the ectoderm will 

 be cleanly cut. In a similar manner, make a parallel vertical cut beneath somite #5 

 at the posterior limit of the pronephric bulge. Then cut the third side of the 

 square between the two ventral points of needle Insertion. This will provide a flap 

 of ectoderm which can be worked away from the neighboring tissues but with the under - 

 lying mesoderm attached . Finally, make the dorsal cut to free the graft and quickly 

 transfer It (always under water) on the tip of a glass needle, Into the previously 

 made hole In the host. Note the orientation of the graft with respect to its origi- 

 nal axes. It may be necessary, to further enlarge the hole of the host, due to heal- 

 ing movements while preparing the graft. Gently press the graft Into place with the 

 hair loop and cover it with a piece of (chipped) cover slip or glass bridge. If the 

 depression is properly made, the cover slip edges will rest on the Permoplast and 

 its center will continually press the graft into place. Do not distort the host by 

 excessive pressure. 



6. After about haJf an hour gently remove the cover slip bridge and allow the 

 embryo to adjust to the new situation. If the graft has not taken, replace the 

 bridge. After another half hour gently shake the embryo from Its depression, and 

 transfer It to a #2 Stender (preferably with agar base) for further growth. If 

 there are loose cells about the wound, clean them away with the hair loop. 



Variations 'In the above procedure would include transplanting the limb ectoderm 

 above or exchanging the limb ectoderm indifferent belly ectoderm before transplant- 

 ing the whole anlage, to determine the place of limb mesoderm In limb determination. 



Eecord by drawings or photographs the condition of the graft at the time of the 

 transplantation and during subsequent weeks. Xenoplastlc transplants between 

 A. punctatum and A. tlgrinum are very instructive. (See section on Limb Field Opera- 

 tions for further details.) 



THE GILLS 



The external gills of the Anura appear as anlagen at 

 stage #l8 and in the Urodela at stage #26. In the Anura 

 they develop as branched, and filamentous outgrowths by 

 stage #22 and in the Urodela by stage #^^1 they are fully 

 formed. Before operating it is well to become fully ac- 

 quainted with the normal morphology and development of 

 the external gills. 



All three germ layers contribute to the formation of 

 the external gills and Harrison (1921) has shown that the 

 gills of Amblystoma punctatum are determined by stage #21, 

 (see also Severinghaus, 1950 and Eotmann, 1935). Trans- 

 plants can be varied to check the relative place of at 

 least the ectoderm and the endoderm, It being rather dif- 

 ficult to Isolate the intermediate mesoderm for such an 

 experlpiental test, although it can be done. 



1. Select two embryos, Anura or Urodela, of the same 

 age and size and place them in Standard Solution 

 in a Permoplast or paraffin operating Syracuse dish. 



Amblystoma tlgrinum gill 

 anlage transplanted to 

 the belly region of A. 

 punctatum. 



Prepare shallow depreesiona 



to hold thnm right side uppermost. Eemove all the membranes. 



