AN ANALYSIS OF THE DYNAMIC FACTORS RESPONSIBLE FOR THE PHENOMENON OF PIGMENT SUPPRESSION IN SALAMANDER LARVAE

¹ School of Biological Sciences, Stanford University, and Department of Zoology, University of North Carolina, Chapel Hill, N. C.

The present study was undertaken for the purpose of clarifying the developmental mechanics operating in the phenomenon of pigment suppression in salamanders. The experiments include the transplantation of epidermis and the explantation, transplantation and extirpation of neural folds and neural crest in five species of salamanders. These species, listed in the order of increasing rates of development, are: Triturus rivularis, T. torosus, Amblystoma punctatum, A. mexicanum and A. tigrinum.

1. By culturing two fragments of neural crest of different age in single drops of culture medium, it was demonstrated that incompletely differentiated melanophores offer a more effective barrier to the migration of young pro-pigment cells than do the cells of fully mature outgrowths.

2. The same relationship between intermediate stage of differentiation and maximal ability to prevent invasion by younger cells was demonstrated in vivo by transplanting young A. punctatum neural crest on the flanks of T. rivularis embryos of graded age.

3. It was found that the degree of pigment suppression obtained in "standard" chimeric-crest embryos (unilateral orthotopic grafts of trunk neural folds) could be reduced either by prolonged chilling of the embryos following the operation, or by using donor and host neurulae of different developmental age. It is assumed that in both instances, the normal difference in donor and host developmental rates was altered in such a manner that an optimal intermediate disparity was not realized.

4. Chimeric-crest taken from A. mexicanum T. torosus embryos (a combination in which suppression is maximally expressed) and explanted in peritoneal fluid gave mixed outgrowths of A. mexicanum and T. torosus cells, both types of which eventually became pigmented. This result is noteworthy, inasmuch as had they developed in vivo, the T. torosus cells undoubtedly would have been suppressed.

5. Amblystoma epidermis which is "strongly melanogenic" was grafted on the flanks of chimeric-crest hosts as a means of testing for the presence of unpigmented T. torosus cells on the flanks. T. torosus melanophores did differentiate under epidermal flank grafts on A. punctatum T. torosus hosts (a combination exhibiting a low degree of suppression), but they failed to appear in A. mexicanum T. torosus hosts except under special circumstances. It is concluded that A. mexicanum cells had prevented the outgrowth and differentiation of T. torosus pro-pigment cells.

6. Homoplastic transplantations of belly epidermis from T. torosus donors of graded age mid-dorsally onto tail-bud hosts revealed that between Stages 38 and 40 +, T. torosus epidermis loses most of its capacity to promote pigmentation in Triturus melanophores. It is suggested that this may be responsible for secondarily delaying the differentiation of T. torosus pro-pigment cells after the donor cells are well differentiated on chimeric-crest hosts.

7. Observations on the manner in which trunk regions deprived of neural crest became invaded by melanophores indicate that melanophore differentiation is essentially a continuous process in T. rivularis, A. mexicanum and A. punctatum embryos. In T. torosus and A. tigrinum embryos, the sequence of pigmentation was found to he interrupted, and consisted of distinct "primary" and "secondary" generations of melanophores.

8. It was noted that the removal of one trunk neural fold from T. rivularis embryos had no observable effect upon the number of pigment cells that later appeared. In similarly treated T. torosus embryos, however, this resulted in a 30 to 40 per cent reduction in the number of cells normally appearing. The results of previous chimeric-crest experiments (Lehman, 1950) are analyzed in the light of the above findings.