TRANSPLANTATIONS 259 



THE BALANCERS 



Balancers are paired, alender, rod-like appendages which project from the side of the 

 head slightly behind and helow the level of the eyes. They are found in Triturus and in 

 Amhlystoma, (except for A. tigrinum), hut never in Bana. They serve as supports which hold 

 the head off the bottom, preventing the larva from losing its balance before the develop- 

 ment of functional forelimbs. They consist of an epithelial membrane whose glands secrete 

 a mucoid substance at the tip and a mesodermal central core with a nerve and blood vessels 

 (see Bodenstein, 19it-5). They normally develop at about stage #^h but the anlage appear as 

 early as stage #21-22 (Bodenstein, I9U5). 



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

 branes. Place them in a Syracuse operating dish with Permoplast base and partial- 

 ly 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 

 embryos. 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 

 forvard, beneath and pai-allel to the ectoderm; allowing the needle to emerge at 

 the upper level of the pre-choeen 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. Bepeat this procedure along 

 the parallel line of the pre-chosen area, then across the ventral and finally the 

 dorgal edge. Lift out this ectoderm and discard. Excavate some underlying meso- 

 derm. 



3. Preparation of transplant. The balancer site is on the mandibular arch. Just pos- 

 terior to and slightly ventral to the eye. The dorsal limit of the balancer an- 

 lage 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 

 ectoderm 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 

 transplant 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 more) 

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

 sterile agar base. Place at a cool ( l8°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 

 determination of the axes at the time of transplantation. Since a balancer does not nor- 

 mally develop on A. trigrinum, an heteroplastic transplant from siny other species to 

 A. tigrinum should be atten^Jted. 



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. In the space on the 

 following page make sketches of each (donor and host) immediately after this operation and 



