Vertebrates 



to cover the limb mesoderm of A. tigrinum 

 the result is a smaller than normal limb. 

 When the chimaeric larval limbs are am- 

 putated they regenerate according to the 

 growth rate of the original mesodermal 

 components. If the fast- or slow-growing 

 species epidermis was an essential part in 

 the formation of the blastema, it did not 

 register in the size of the regenerate although 

 it did have an effect upon the original limb 

 growth. This indirect evidence suggests 

 strongly that there is no epidermal contri- 

 bution to the blastema. 



Another line of evidence for the impor- 

 tance of mesoderm in the formation of the 

 blastema is found in the work of Karczmar 

 and Berg ('51). They have studied the occur- 

 rence of alkaline phosphatase during both 

 embryonic development and regeneration in 

 Amblystoma. Brachet ('46), Krugelis ('50), 

 Lindeman ('49) and Moog ('44) have cor- 

 related the alkaline phosphatase content 

 with various steps in differentiation phases 

 of normal ontogeny. The importance of the 

 study is that it tests whether normal onto- 

 genic processes, in which alkaline phospha- 

 tase levels are low, are duplicated in the 

 dedifferentiation subsequent to injury. 



Histochemical localization of alkaline 

 phosphatase was made ( 1 ) on 25 young larvae, 

 13 to 17 mm. long, (2) on 60 regenerating 

 larvae, 25-47 mm. long, and (3) on adult 

 regenerating limbs. In ontogeny the hind 

 limb primordium (stage 47) shows cells with 

 a high alkaline phosphatase content. As 

 the limb bud (stages 49 and 50) begins to 

 project from the body wall, the phosphatase 

 activity is strongest at its apex. This relation 

 persists and increases as the limb parallels 

 the trunk (stage 50); later (stage 50-f-) the 

 stain shows a definite concentration at the 

 base of the elongating limb, marking the 



683 



site of presumptive limb components. In 

 stages 51 and 52, enzyme activity decreases 

 locally and the differentiating tissues sepa- 

 rate regionally into loci of lower and higher 

 enzyme activity. 



This brief description is applicable to the 

 regional development of the differentiating 

 limb components. When, however, the spe- 

 cific tissue differentiations occur there is a de- 

 crease in the alkaline phosphatase activity 

 during myogenesis and by the time the muscle 

 bundles are formed the phosphatase de- 

 creases to the relatively low larval level. A 

 similar condition prevails in cartilage for- 

 mation, although there is a slight but 

 perceptible rise at the beginning of bone 

 differentiation. Throughout limb develop- 

 ment, there is no demonstrable localization 

 of alkaline phosphatase in the epidermis of 

 the body wall. 



Karczmar and Berg ('51) (see photomicro- 

 graphs 1 to 7, Fig. 239) have divided the 

 regeneration process into three overlapping 

 phases: (1) dedifferentiation, (2) growth and 

 (3) differentiation. They have traced the 

 localization of the alkaline phosphatase in 

 each of these stages. There is an increase in 

 level about three days after amputation, 

 rising through the growth period between the 

 fourth and fifth day and becoming region- 

 ally localized in the regenerate on the fif- 

 teenth day (see photomicrograph 7, Fig. 

 239). After this the enzyme distribution 

 follows the ontogenetic pattern. 



The evidence on the epidermal contribu- 

 tion to the blastema seems clear. The epi- 

 dermis covers the wound in a few hours, but 

 is phosphatase-negative until six days after 

 amputation, by which time the blastema is 

 well formed and growing. The localization 

 of the enzyme is variable, after this period 

 stronger at the apex of the epidermis over 



structures still provide an accurate measure of enzyme activity: muscle stains + (single plus); acellular 

 diaphyseal shaft + -f (double plus). Blastematic nuclei (B) stain -|- + -j- (triple plus). X 20. 



5, Conical blastema, staining -{- -f + (triple plus), 10 days after amputation. This section through 

 the margin of the perichondrial sheath of the humerus, P -\- -\- (double plus) demonstrates the enzyme 

 distribution in muscle. H, Hypodermis, + + + (triple plus); D, enzyme-free distal edge of collagenous 

 derma, -f- (single plus) marking the level of amputation; Ch, hyaline cartilage, with -j- (single plus) 

 matrix. Note the patches of enzyme activity on the epidermis. X 20. 



6, Differentiating blastema with regions of high and low enzyme activity, 13 days after amputation. 

 D, Newly secreted matrix of the differentiating derma, -|- (single plus); Pu, presumptive perichon- 

 drium, + + + (triple plus) of humerus and ulna — the parallel, -f + (double plus) area is the 

 differentiating muscle of upper arm and forearm; B, finger-bud blastema, + + -]- (triple plus). X 80. 



7, Detail of a differentiating blastema, 12 days after amputation. C, Rudiment of humerus, illustrating 

 from right to left, the primary differentiation of chondrocytes accompanied by a drop in enzyme activity 

 from + + -f (triple plus) to -f (single plus), and the secondary increase of intra- and extra-cellular 

 enzyme activity in the hypertrophic chondrocytes; M, differentiating muscle, staining -\- -f (double 

 plus); P, differentiating perichondrium, staining -f + -f- (triple plus); V, blood vessel with + + + 

 (triple plus) walls. 



