5lU 



REGENERATION 



THE DEVELOPMENT OF ORGAN ANLAGEN IN REGENERATING BLASTEMAS 



This experiment Involves regeneration, transplantation, and possibly some Induction. 

 It is based upon the assumption that the regenerating tissue (the blastema) is essentially 

 embryonic in nature and will either supplement an implanted organ anlage or will, under 

 tJie influence of such an anlage, bs induced to form certain organs. 



1. Select 20 to 50 frog tadpoles measuring at least 3.5 cm In total length. They 

 may measure as much as 8 cm. The younger ones are generally better, even though 

 the blastema is, in consequence, smaller. 



2. Anesthetize the tadpoles with 1/10,000 MS 222 and, with a sharp scalpel, cut 

 off the tail fin at an angle toward the dorsal side of the body about 5 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 ex- 

 act date. 



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

 tails. After about 5 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 ^astrula 

 to be implanted into the blas- 

 tema, 



(From Emerson 19^+1: 

 Jour. Exp. Zool. 8T:'+03) 



Regions of the late gastrula to 

 be implanted into the blastema, 

 M - the anterior limit of the 

 presumptive neural plate, 



( From Emerson 19i^2 : 

 Jour. Exp. Zool. 90:553) 



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

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

 generally the most satisfactory, and these may be taken from late tall-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 



Forebraln vesicle. 



Medullary plate or neural fold. 



Hypophysis 



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

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

 lancet (or Iridectony scissors) gently cut between the blastema and the old 

 tissue (of the tall) along the side of the tadpole. Then make two shallow 

 cute, 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. 



