262 TRANSPLANTATIONS 



1. Select two Urodele embryos of about stage #28 and shell them out of their membranes. 

 Place them in a Syracuse operating dish with Permoplast base and partially filled 

 with Urodele Operating Medium. Prepare depressions with ball tip, side-by-side and 

 just adequate for the embryos. 



2. Prepare the host embryo. Choose the site for the transplantation on one of the em- 

 bryos. The most satisfactory positions are just posterior to the pronephros and in 

 line with it, or just posterior to the otocyst. With glass operating needles cut out a 

 rectangular piece of ectoderm about the size of two somites. This may be done by 

 piercing the ectoderm with the point of the needle; pushing the needle forward, beneath 

 and parallel to the ectoderm; allowing the needle to emerge at the upper level of the 

 pre-chosen area, and then (if the needle is rigid) lifting it upward and thereby making 

 a clean cut. If this seems impractical, rub a hair loop against the needle until the 

 ectoderm is cut. Repeat this procedure along the parallel line of the pre-chosen 

 area, then across the ventral and finally the dorsal edge. Lift out this ectoderm and 

 discard. Excavate some underlying mesoderm. 



3. Preparation of transplant. The balancer site is on the mandibular arch, just poster- 

 ior to and slightly ventral to the eye. The dorsal limit of the balancer anlage is on a 

 level with the dorsal limit of the eye and the ventral limit on a line below the ventral 

 limit of the eye. Anteriorly the balancer area touches that of the eye and posteriorly 

 it is below the second gill slit. 



In a manner similar to that of host ectoderm removal (above) remove the balancer ecto- 

 derm from the area indicated and on the tip of a needle (or hair loop) transfer it to the 

 prepared (excavated) site on the host. Try to retain the same orientation of the trans- 

 plant in the new, host environment, in respect to the original antero-posterior , dorso- 

 ventral axes. (Do not rotate the transplant. ) If the host implantation site has closed over 

 in the interim, remove some mesenchyme cells with the hair loop and widen the hole to 

 fit the transplant. Work as quickly as possible since the transplant is apt to fall apart. 

 As soon as the transplant is in position, press it gently into place with the hair loop and 

 lay over it a piece of coverslip (which will act as a bridge). The embryo should be left 

 undisturbed for at least 30 minutes, in a cool, dark place. 



After about 30 minutes if it seems that the transplant is not "taking", or has been moved, 

 scratch the host site with a needle, replace the transplant and bridge, and await further 

 healing. When the transplant is definitely attached, gently remove the bridge and allow 

 the embryo to adjust to the new situation for a few minutes. Work the embryo out of its 

 depression, by means of hair loops, shake it free, and (after about 1 hour or nnore) re- 

 move it with a large-mouthed, clean pipette to a #2 covered Stender provided with a 

 sterile agar base. Place at a cool (18°C. ) temperature. 



It will be instructive to make the transplant between individuals of different ages, and 

 also to rotate the transplant 90° or 180° in the host site to determine the degree of de- 

 termination of the axes at the time of transplantation. Since a balancer does not normally 

 develop on A. trigrinum, an heteroplastic transplant from any other species to A. tigri- 

 num should be attempted. 



Both donor and host may be kept in the same Stender, and the balancer site and anlage' of 

 the other (bilateral) side of each may be considered as control. 



Make drawing or photographic records of individuals at appropriate intervals. 



