LIMB FIELD OPERATIONS 



[anlage) of the 



PURPOSE : :^y the application of experimental methods to the limb field 

 Urodele embryo, to determine the 



a. Powers of regeneration following partial and total extirpation of the anlage. 



b. The effect of rotating the limb field axis. 



c. The effect of splitting the limb field. 



d. The relative f mictions of the ectoderm and the mesoderm in limb formation. 



e. The differentiating capacity of heterotopic transplants. 



f. The relation of host and donor in xenoplastic transplants. 



g. The genetic factors relating to limb formation, by means of 



1. Heterogenic transplants (i.e., between species with different rates 

 of development. 



2. HJsteroplastic transplants involving pigmentary differences. 



MATERIALS : 



Biological : Urodele larvae of stages #29 to #55. 



Slow developing forma 



Amblystoma punctatum ( maculatum) 

 Amblystoma texanum (mlcrostomum) 

 Amblystoma Jefferaonianum 

 Amblystoma opacum 

 Triturus rivularls 

 Tritunis slmulana 

 Trdturus torosus 



Bapid developing forma 



Amblystoma tlgrlnum 

 Amblystoma mexicanum (axolotl) 



Technical : Standard Operating Equipment. 



Vital dyes and MS 222 freshly made up cone. l/5,000. 



METHOD : 



Precautions : 



a. Use 0.1^ sodium sulfadiazine if bacterial infection is encountered. 



b. Be reluctant to examine the transplant. It should remain undisturbed for 50 

 minutes or more and any necessary transferring should be by wide-mouthed pipette. 



c. Temperatures lower than that of the laboratory are best for Urodele recovery and 

 survival. Some species do best at 10°C. 



d. Operations are performed in Operating Medium; embryoa are later transferred and 

 raised in Growing Medium. Feeding should begin when the balancers disappear. 



e. Muscular activity begins at about Stage #3**-. If operations are attempted at this 

 stage it will be neceasary to use freshly made MS 222 in operating medium l/5,000. 



Control : The controla for each of the following experimental procedures will, of neces- 

 sity, be different. 



Procedure : Each of the following exercises deals with the limb field but since each 

 illustrates a different principle, the procedures will naturally differ somewhat. 



REGENERATION FOLLOWING EXTIRPATION OF THE ANLAGE** 



Secure embryos at stage #29 and remove all membranes. Place the embryos in a Petri 

 dish with an agar base. Select those which are exactly at stage #29 and compare with the 

 accompanying diagram on following page. 



* The author acknowledges, with appreciation, the help of the late Dr. F. H. Swett in 

 organizing this exercise. 

 ** Limb extirpation la included in the exercise on "Extirpations" but the procedure here 

 Is considerably more detailed. 



-21k- 



