270 ORIGIN OF AMPHIBIAN PIGMENT 



(d) Transfer for one-half hour to a mixture of equal parts of 100% alcohol, (contain- 

 ing 0. 1% phosphomolybdic acid) and cedar oil. Then place in pure cedar oil until 

 clear (overnight). 



(e) Embed in three changes of paraffin-Bayberry-beeswax mixture (90 - 5 - 5) and, 

 when hardened, section at 10 microns. The sections may be treated in the usual 

 way (with xylol) and mounted under clarite. If the tissues are not passed through 

 any water they will retain the Nile Blue Sulphate dye in the cells which were or- 

 iginally stained, and with appropriate lighting the demarcation between stained 

 and unstained areas can be made out easily. 



OBSERVATIONS AND TABULATION OF DATA: 



The data for these experiments are qualitative rather than quantitative. Histological 

 sections, when possible, will confirm the macroscopic analyses. (See DuShane, 1935: 

 Jour. Exp. Zool. 72:1 for cytological procedures relative to the various types of chrom- 

 atophores. ) 



DISCUSSION: 



The neural crest arises as a strip of cells lying between the neural plate and the dorsal 

 ectoderm, this plate being separated from both of these structures during the closure of 

 the neural folds. It is now established that the neural crest is the principal and probably 

 the sole source of all pigment cells (except the tapetum) in all of the vertebrates that have 

 been studied (DuShane, 1943). Melanophores are found in the epidermis and dermis, 

 meninges, visceral mesenteries, peritoneum, and in close association with the blood ves- 

 sels throughout the body. This means that from the original source of such pigment cells 

 there has been very extensive migration. The forces involved in this migration are illus- 

 trated in the capillary-tube isolation experiments of Twitty (1944). But the pre-migratory 

 neural crest cells cannot be distinguished from their neighbors because they show no pig- 

 ment differentiation until about the time they reach their normal destination. Such pig- 

 ment cell differentiation includes: 



Melanophores - wide distribution, cells with brown to black melanin. 



Lipohores - dermis and epidermis, having diffuse yellow pigment (lipochrome) in 



solution. 

 Guanophores - pericardium and most lateral line organs, highly refractive, granular, 



golden yellow guanin crystals with metallic lustre. 



The pigment pattern may be used to identify different species of Amblystoma or Triturus 

 and frequently in heteroplastic transplantations there is evidence that both genetic and 

 environmental factors (such as humoral or contiguous cell influences) may be important 

 in directing ultimate cellular differentiation. In Amblystoma punctatum there is even dis- 

 tribution of melanophores while the lipophores are fused to give a continuous sheet within 

 the dermis. In A. tigrinum the melanophores are large and dark and are arranged in 

 groups, as are also the lipophores. The time of melanophore appearance is species spe- 

 cific. In A. punctatum the first melanophores appear at stage #34 lateral to the nneduUa, 

 beneath the epidermis, and by stage #36 they have reached the level of the pronephros. 

 The lipophores begin to appear at stage #28. Potential melanophores from various spe- 

 cies manifest different abilities to develop in isolation, and even in the normal environ- 

 ment there are melanophores which are dependent upon the presence of pigmented epider- 

 mis to produce melanin. Other derivatives of the neural crest may include chromaffin 

 tissue, mesenchyme, sheath cells, visceral cartilages, spinal ganglion (neuroblast) cells, 

 sympathetic ganglia, and adrenal medulla. 



