258 TRANSPLANTATIONS 



2. Select a pair of embryos of similar developmental stage and place them side-by-side 

 in an operating dish over soft paraffin or Permoplast. Mould a depression for the 

 host, and place it in the depression with the right side uppermost. 



3. Locate the forelimb anlage. This will be found ventral to somites #3-#5, just poster- 

 ior to the gill swelling and includes a portion of the ventral slope of the pronephric 

 bulge. 



4. Prepare the host by cutting a square hole with a glass needle (or a lancet) about 3 

 somites in diameter at the level of the pronephric bulge but just beneath the somites 

 #8 to #10. The excavation must be deep enough to include some underlying meso- 

 derm. 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 be- 

 neath 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 underlying 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 original axes. It may be necessary 

 to further enlarge the hole of the host, due to healing 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 half 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 an- 

 other 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 transplanting 

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



Record by drawings or photographs the condition of the graft at the time of the trans- 

 plantation and during subsequent weeks. Xenoplastic transplants between A. punctatum 

 and A. tigrinum are very instructive. (See section on Limb Field Operations for 

 further details. ) 



THE GILLS 

 (See also page 251) 



The external gills of the Anura appear as anlagen at stage #18 and in the Urod.ela at 

 stage #26. In the Anura they develop as branched, and filamentous outgrowth by 

 stage #22 and in the Urodela by stage #41 they are fully formed. Before operating it 

 is well to become fully acquainted 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, 1930 and Rotmann, 1935). Transplants can be varied to check the relative 

 place of at least the ectoderm and the endoderm, it being rather difficult to isolate the 

 intermediate mesoderm for such an experimental test, although it can be done. 



