666 



Regeneration 



ance. This idea is best illustrated in the 

 experiments dealing with recombination of 

 animal with vegetal blastomeres in the sea 

 urchin egg (Horstadius, '35). 



In any case, one of the real problems for 

 future work is that of deriving multiple 

 differentiations from one or even two gra- 

 dients. It is evident that, if the presumptive 

 head of a regenerate contains the same sub- 

 stance or substances as the presumptive tail, 

 some kind of quantitative thresholds for 

 head and tail formation must be assumed. 

 Since the regenerate already contains a va- 

 riety of cell types which may possibly react 

 in the gradient system by differentiating 

 into a variety of structures, the problem of 

 deriving multiplicity from a single gradient 

 may be easier than first appears. 



THE STIMULUS 



The egg responds to a number of stimuli 

 by beginning to develop. One theory of 

 stimulation states that detoxification of the 

 unfertilized egg results. The egg is pictured 

 as being inhibited by toxic products which 

 escape or are destroyed at fertilization. What 

 stimulates tissues of the adult to differen- 

 tiate? Cutting and injury serve as the im- 

 mediate stimulus and the problem becomes 

 one of determining just what cutting does to 

 tissues. 



In this connection, consider the possibility 

 that it is not the cutting or injury per se 

 but rather the isolation of tissues from cor- 

 relative influences of adjacent tissues that 

 is the effective stimulus for differentiation. 

 For example, the normal growth form of 

 the ascidians makes this possibility very 

 probable (Berrill, '51). Perophora reproduces 

 by budding from the stolon. The stolon 

 grows along a surface in a straight line 

 away from the zooid. At fairly regular space 

 intervals along this growing stolon, buds 

 appear and differentiate into zooids. This 

 observation suggests that the tissues of the 

 growing stolon are inhibited from forming 

 buds until the stolon reaches a length at 

 which its tissues are below the threshold 

 for inhibition by the zooid. A bud then 

 forms which develops into a zooid. The 

 stolon continues growing and this new de- 

 veloping zooid exercises an inhibition over 

 the tissues of the stolon until the latter 

 reach a certain distance, when again the 

 inhibition falls below a threshold and a 

 second bud forms. The important fact is 

 that no cutting or external stimulus needs 

 to be applied for the formation of a bud. 



Therefore when a section of the stolon is 

 cut and removed from a colony the major 

 effect may be the removal of the inhibition 

 exercised by the zooid and not the stimulus 

 of cutting or injury. In this connection, 

 recall Huxley's experiments (Huxley, '26) 

 in which the equilibrium, stolon ^ zooid, 

 was controlled by the addition of external 

 agents such as potassium cyanide and po- 

 tassium chloride. 



On the other hand the differentiation of 

 zooids from the stolon always occurs at the 

 cut ends and not from the middle of an 

 excised section. This observation shows that 

 the cut end is stimulated in some way. Thus 

 at least two dissociable factors operate in 

 the stimulation of regeneration: (1) correl- 

 ative factors, i.e., the removal of inhibitory 

 regions, and (2) a direct stimulus caused by 

 cutting or injury. The correlative factor will 

 be discussed later and at this point we will 

 proceed to analyze the stimulus of cutting. 



A number of observations demonstrate 

 conclusively that cutting tissues does not 

 suffice for regeneration unless the wound 

 remains open to the external medium. In 

 Tubularia a hydranth fails to regenerate 

 at a cut end if the perisarc covers the surface 

 of the wound (Barth, '40). Likewise in the 

 ascidian stolon when the cut is made with 

 a blunt scissors the tunic is pinched together 

 and a zooid fails to form. The perisarc and 

 tunic act as barriers to regeneration (Goldin, 

 '48). Furthermore, a cut surface is not 

 necessary for regeneration of a hydranth in 

 Tubularia. If the perisarc is removed from 

 the middle of an isolated piece of stem, 

 regeneration of a hydranth will occur from 

 the intact uncut stem (Zwilling, '39). 



The general conclusion from these ex- 

 periments and observations is that a layer 

 at the surface of tissues prevents regenera- 

 tion. In Tubularia and ascidians the layer 

 is identified as perisarc and tunic, respec- 

 tively, but in other forms we may assume 

 the presence of some similar limiting layer. 

 In amphibians this layer is a component of 

 the skin (Godlewski, '28; Rose, '44) which 

 inhibits regeneration of limb and tail. 



How does this external sixrface layer act 

 to prevent regeneration? Experiments on 

 Tubularia with vital dyes show that neutral 

 red penetrates rapidly into cells at an ex- 

 posed surface but very slowly into cells 

 covered with the perisarc. Thus the perisarc 

 acts as a barrier to free diffusion of neutral 

 red and perhaps other substances. Now it is 

 known that the excretory products of Tubu- 

 laria inhibit regeneration completely. A glass 



