Isolation of a Factor Mediating Melanogenic Induction in Pigment Cells of Xenopus Laevis

An activity has been identified in +/+ Xenopus laevis embryonic extract which stimulates intensive and stable melanoprotein synthesis in retinal pigmented epithelium and dermal melanophores of albino periodical mutants, a^p/a^p. Experiments involved four steps: 1) preparation of extract from +/+ embryos at stages NF 18–21 when melanogenesis occurs most extensively; 2) gel-filtration of the extract; 3) microinjection of the fractionated materials into a^p/a^p; and 4) identification of the nature of active substances. The activity is heat-labile and is destroyed by treatment with pronase E. We call this material melanogenic factor (MGF) and suggest that it is responsible for initiating melanogenic activity in melanin-synthesizing cells.     

Mechanisms of Melanophore Induction in Amphibian Development

Induction of melanophores was examined by the sandwich method of explantation with embryonic tissues of Xenopus laevis +/+ and the white mutant, a^P/a^P. Interspecific combinations of tissues of Triturus taeniatus and Xenopus borealis were also used. The ectoderm used as the reacting system was taken from embriyos at various stages and combined with various tissues known to be melanogenic inductors. The following results were obtained: 1) The sources of melanophore induction in both +/+ and a^p/a^p studied by sandwich explantation were the same in both retinal pigmented epithelium and dermal melanophores: 2) Melanophores were induced in epidermal material from embryos at stages from the early gastrula to the late tail bud stage: 3) The presence of melanoblasts together with other ectomesenchymal cells in the neural crest is not sine qua non for their determination and differentiation: 4) On isolation of reacting material from the late gastrula, melanophores appeared in all cases. This shows that two hours contact between inductor tissues and the ectoderm is necessary and sufficient for melanophore induction: 5) Melanophore induction is not species-specific, but occurred in Xenopus ectoderm under the action of endomesoderm of Tr. taeniatus or X. borealis , and vice versa. The shapes and structures of melanophores induced were typical for the species from which the ectoderm was taken: 6) Melanogenic activity in the late gastrula stage has a gradient of distribution with a maximum in the prechordal plate: 7) In the mutant only the primary source of melanogenic inductors, the prechordal plate (PrP1), was active in stages both before and after its invagination: 8) Despite the fact that skin melanophores and retinal melanocytes have different genesis in development, all the present data suggest the identity of the mechanisms of melanin synthesizing machinery in the two.     

On the Melanization of the Rat's Eye

Electron-microscopic study of the size of the melanosomes, the mean percentage of melanosomal profile area (MPMA) of the cells, and the duration of melanogenesis in the pigmented layers of the rat's eye (inbred strain BDE/Han) revealed the following: 1) The melanosomes in the cells of the retina vary in size and shape in different locations of the eye. The MPMA of the cells also differs. Only in the two layers of the iris epithelium do the minor diameters of the melanosomes not differ significantly from each other, but the MPMA of the cells is different. The pigmented outer layer of the ciliary epithelium stands out on account of its especially large, round melanosomes. 2) The melanosomes of the uveal melanocytes are uniformly small but exhibit the largest MPMA. 3) Only in the pigment epithelium of the fundus does melanogenesis cease in the fifth week of life. As a result the MPMA decreases. In the other areas of the pigmented epithelium and the uvea tyrosinase activity and premelanosomes are present from the new-born to the adult animal. These signs indicate continued melanogenesis. 4) Compound melanosomes are present in all pigmented locations of the eye. Giant melanosomes occur regularly only in the outer layer of the retina.     

The effect of 5-bromodeoxyuridine on the differentiation of chick embryo pigment cells

Cultures of (a) dispersed presumptive melanoblasts from chick somites and (b) organ cultures of retinal melanoblasts were grown in control medium and medium containing 5-bromodeoxyuridine (BUdR). The somite cell cultures at time zero had no evidence of melanogenic organelles; the eye cultures, from embryos of the same stage, stage 16–18, contained cells showing the initial stages of melanogenesis. In the presence of BUdR, presumptive trunk melanoblasts failed to pigment, while controls became heavily pigmented, whereas cells in the retinal pigment epithelium made melanin but in reduced amounts when compared with controls. These results suggest different methods of control over synthetic programs depending upon whether synthesis has, or has not, been initiated when the cells are exposed to BUdR.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Mutant gene expression in mouse aggregation chimeras. 8. The effect of the white gene on coat pigmentation

Aggregation of mouse embryos produced 11 chimaeras Miwh/+C/C----+/+c/c and 8 chimaeras +/+C/C----+/+c/c (control). Chimaerism was detected by mosaicism of coat retinal pigment epithelium and by electrophoretic pattern of glucose phosphate isomerase. All chimaeras showed a common pattern of pigmented and unpigmented hair regions that alternated as stripes of different length and width and extended from spine in lateral-ventral direction. However, white coat color predominated in Miwh/+C/C----+/+c/c chimaeras due to a higher proportion of unpigmented zones as well as to weakening of hair color in pigmented areas. Besides, distal regions of limbs were always unpigmented in Miwh/+C/C----+/+c/c chimaeras and completely or partially pigmented in +/+C/C----+/+c/c chimaeras. Pigmented hair regions are often located on the ventral trunk surface where the Miwh/+ heterozygotes usually had an unpigmented spot. The examination of hairs, taken from the same regions of gray coloration, revealed the presence of pigmented, unpigmented and mosaic hairs. The proportion of unpigmented hairs was much higher in Miwh/+C/C----+/+c/c chimaeras than in +/+C/C----+/+c/c chimaeras. The data obtained indicate that a single Miwh gene dose reduced proliferative activity of melanoblasts which resulted in weakening of coat pigmentation.     

The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis

Summary The crystallins of normal and a^p mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (a^p/a^p; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.     

Vacuolar-type H<Superscript>+</Superscript>-ATPase with the <Emphasis Type="Italic">a</Emphasis>3 isoform is the proton pump on premature melanosomes

The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H⁺-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1–a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes. Ge-Hong Sun-Wada and Yoh Wada contributed equally to this study. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi and Noda Foundations.     

Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates.

The amphioxus tail bud is similar to the amphibian tail bud in having an epithelial organization without a mesenchymal component. We characterize three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) and show that their early expression around the blastopore can subsequently be traced into the tail bud; in vertebrate embryos, there is a similar progression of expression domains for Wnt3, Wnt5, and Wnt6 genes from the blastopore lip (or its equivalent) to the tail bud. In amphioxus, AmphiWnt3, AmphiWnt5, and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis. In spite of similarities within their tail buds, vertebrate and amphioxus embryos differ markedly in the relation between the tail bud and the nascent somites: vertebrates have a relatively extensive zone of unsegmented mesenchyme (i.e., presomitic mesoderm) intervening between the tail bud and the forming somites, whereas the amphioxus tail bud gives rise to new somites directly. It is likely that presomitic mesoderm is a vertebrate innovation made possible by developmental interconversions between epithelium and mesenchyme that first became prominent at the dawn of vertebrate evolution.     

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin‐containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross‐talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus‐mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication‐Competent Avian sarcoma‐leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back‐transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)⁺Neo^r+‐electroporated melanocytes from Hypeco nera were selectively grown in G418‐supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS‐mOrange, mOrange⁺ keratinocytes and transplanted EGFP⁺ melanocytes were frequently juxtaposed each other. High‐resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.     

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.     

Interaction of the Murine Dilute Suppressor Gene (Dsu) with Fourteen Coat Color Mutations

The murine dilute suppressor gene, dsu, was previously shown to suppress the dilute coat color phenotypes of mice homozygous for the dilute (d), leaden (ln), and ashen (ash) mutations. Each of these mutations produce adendritic melanocytes, which results in an abnormal transportation of pigment granules into the hair shaft and a diluted coat color. The suppression of each mutation is associated with the restoration of near normal melanocyte morphology, indicating that dsu can compensate for the absence of normal d, ln and ash gene products. In experiments described here, we have determined whether dsu can suppress the coat color phenotype of 14 additional mutations, at 11 loci, that affect coat color by mechanisms other than alterations in melanocyte morphology. In no case was dsu able to suppress the coat color phenotype of these 14 mutations. This suggests that dsu acts specifically on coat color mutations that result from an abnormal melanocyte morphology. Unexpectedly, dsu suppressed the ruby eye color of ruby-eye (ru) and ruby-eye-2 (ru-2) mice, to black. The exact nature of the defect producing these two mutant phenotypes is unknown. Histological examination of the pigmented tissues of the eyes of these mice indicated that dsu suppresses the eye color by increasing the overall level of pigmentation in the choroid but not the retinal pigmented epithelium. Choroid melanocytes, like those in the skin, are derived from the neural crest while melanocytes in the retinal pigmented epithelium are derived from the optic cup. This suggests that dsu may act specifically on neural crest-derived melanocytes. These studies have thus identified a second group of genes whose phenotypes are suppressed by dsu and have provided new insights into the mechanism of action of dsu.     

Modification of pigmentation patterns in allophenic mice by the W gene

Mouse embryos heterozygous at the W locus were combined with embryos which were wild type at this locus but homozygous for albino. The resulting allophenics displayed an unusual pigmentation phenotype consisting of entirely white fur and ruby-coloured eyes. Microscopic examination showed the eye pigment to be located exclusively in the retinal epithelium, which was a mosaic of black and white sectors. This ruby-eyed white pattern corresponds to what would have been expected for WWCC in equilibrium wwcc mosaics but not for WwCC in equlibrium wwcc mice. WW mice are black-eyed whites, but Ww mice have black eyes and black fur, except for a small ventral white spot. These results suggest that melanocytes of the Ww genotype, although capable of producting normally pigmented fur in Ww animals, fail to populate hair follicles when the competition with wwcc (albino) melanocytes that are wild type at the W locus. The genotype of these WwCC in equilibrium wwcc alophenes was proved by progeny testing. This is apparently the first report of a single gene change affecting the competitive ability of cells in allophenic mice, and suggests that such changes may play a significant role in the clonal selection of embryonic cells during development.     

Quantification of Ca2+ binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l ‐tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca²⁺ binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (±0.2) × 10³/M. This value is comparable with other well‐established intracellular calcium‐binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca²⁺ displacement assay, the pK_a of the carboxyl group coordinated to Ca²⁺ is determined to be 3.1 ± 0.1.     

Effects of Mutations at the W Locus (c-kit) on Inner Ear Pigmentation and Function in the Mouse

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survivial of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (W^v/W^v) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (W^x, W^sh, and W⁴¹) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of W^sh/W^x, 25% of W^sh/W^sh, 60% of W⁴¹/W^x, and 69.2% of W⁴¹/W⁴¹ ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W⁴¹/W^x, W^sh/W^sh, and W⁴¹/W⁴¹ mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.