No abstract

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 alpha-melanocyte stimulating hormone (alpha-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 less than 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 greater than 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.(ABSTRACT TRUNCATED AT 400 WORDS)

In the periodic albino mutant (a(p)/a(p)) of Xenopus laevis, peculiar leucophore-like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild-type (+/+). These leucophore-like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of alpha-melanocyte stimulating hormone (alphaMSH), and (4) containing an abundance of bizarre-shaped, reflecting platelet-like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore-like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to alphaMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore-like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore-like cells are essentially similar to melanophores of the wild-type with respect to their localization in the skin and manner of response to alphaMSH, (3) the leucophore-like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet-like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore-like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.

That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R. pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15. Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R. pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period. Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation. Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P. dacnicolor in VCM. The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians. The results are discussed in terms of a prevailing theory about pigment cell origins and development.

A ventrally localized melanization-inhibiting factor (MIF) may play an important role in the expression of dorsal-ventral pigment patterns of amphibians. In efforts to purify this putative MIF, ventral skin conditioned medium (VCM) from Rana forreri was partially fractionated and used to immunize mice. A monoclonal antibody that has the ability to block the activity of MIF was isolated, and an immunoaffinity matrix was prepared by cross-linking the antibody to protein G-Sepharose. The fraction of VCM that bound to the affinity matrix decreased the number of melanized cells in the Xenopus laevis neural tube explant assay, but did not reduce significantly the number of cells that emigrated. The monoclonal antibody was used for immunohistochemical studies on R. pipiens skin. Strong staining with the antibody was observed beneath the basement membrane, in mucous glands, and in the subcutaneous tissue of the ventral skin. A weak staining was also observed in the ground substances of both ventral and dorsal skin. These results confirm that a monoclonal antibody has been secured against at least one of the MIF constituents and that it is useful as a probe in detecting the distribution of MIF in tissues. The results of its use in this study support the hypothesis that MIF plays a role in the expression, development, and maintenance of the dorsal-ventral pigmentation patterns of frogs.