REGENERATION 



307 



Select 20 to 30 frog tadpoles measuring at least 3. 5 cm. in total length. They 

 may measure as much as 8 cn-i. The younger ones are generally better, even 

 though the blastema is, in consequence, smaller. 



Anesthetize the tadpoles with 1/10, 000 MS 222 and, with a sharp scfalpel, cut off 

 the tail fin at an angle toward the dorsal side of the body about 3 mm. from the 

 body. The older tadpoles may exhibit some hemorrhage but this can be stopped, 

 if necessary, by brief exposure to hypertonic medium. Record the exact date. 

 Allow these tadpoles (in any community crystallizing dish) to regenerate their 

 tails. After about 3 days (and until about 8 days) there will appear a whitish 

 growth bulging from the cut surface. This is the blastema. 



Regions of the early gastrula 

 to be implanted into the blas- 

 tema. 



(From Emerson 1941: Jour. 

 Exp. Zool. 87:403) 



Regions of the late gastrula to 

 be implanted into the blastema. 

 M - the anterior limit of the 

 presumptive neural plate. 



(From Emerson 1942: Jour. 

 Exp. Zool. 90:353) 



Prepare donor material, consisting of various organ anlagen. Consult the Vogt 

 map of organ fields of early gastrula. The eye and the sucker anlagen are gen- 

 erally the most satisfactory, and these may be taken from late tail-bud stages. 

 A suggested list of satisfactory organ anlagen follows: 



Early and late gastrula ectoderm areas (see accompanying diagrams) 



Optic vesicle, with and without lens ectoderm 



Auditory (otic) vesicle 



Olfactory vesicle 



Sucker 



Gill bud 



Limb bud 



Forebrain vesicle 



Medullary plate or neural fold 



Hypophysis 



Prepare the host as follows: Anesthetize a host (with pronounced blastema) in 

 1/10, 000 MS 222 in Standard Solution or Spring Water. With a sharp-pointed 

 lancet (or iridectomy scissors) gently cut between the blastema and the old tis- 

 sue (of the tail) along the side of the tadpole. Then make two shallow cuts, one 

 along the dorsal and the other along the ventral margins of the blastema. This 

 will form a V-shaped cut with the apex of the "V" at the most posterior limit of 

 the regenerating blastema. The operating area is small and this is a delicate 

 operation. 



Quickly excise the anlage' to be transplanted and insert it beneath this flap of 

 blastema tissue, orienting it so that its ectodermal layer is outermost. Replace 

 the flap of blastema ectoderm and hold it down for 1 to 2 minutes with a ball tip. 

 The sticky bla-stema cells will become attached to the transplant and will hold it 

 in place. The transplant may be fixed any tinie from 18 hours to a month after 

 the transplantation, sectioned and studied to determine the degree of differentia- 

 tion and induction. 



