Notes: The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, and grafts of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.

Notes: A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW 〈30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW 〉 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.

Notes: Three types of pigment cells were isolated and cultured from larval Rana pipiens, and their attachment, maintenance, and proliferation were examined in the presence of extra-cellular matrix constituents (ECMs) in primary cell culture. The initial profile of pigment cell types present on day 2 of culture reflects the relative attachment of the cells to the dishes. Changes in the numbers of cells present after day 2 reflects the influence of factors present in the culture media on the maintenance, proliferation, or detachment of each type of pigment cell. Fetal bovine serum (FBS) promoted melanophore expression, but inhibited iridophore expression. FBS had no effect on xanthophores. In contrast, ventral skin conditioned medium (VCM), which contains melanization inhibiting factor, strongly stimulated iridophore expression, while it markedly inhibited melanophore expression. VCM had little effect on xanthophores. Of the ECMs tested, collagen type I had no effect on pigment cells. Fibronectin slightly inhibited melanophore expression, while it moderately stimulated iridophores and xanthophores. The stimulatory effect of fibronectin was not as strong as that of FBS or VCM. Laminin was also tested; however, it did not allow pigment cells to attach to the dishes, at least under the culture conditions utilized. The results of these experiments are discussed in terms of the general mechanisms of pigment pattern formation.