Dopachrome conversion and dopa oxidase activities in recessive yellow mice. Catalytic activities of extracts from pheomelanic and eumelanic tissues

The dopa oxidase activity of tyrosinase and the dopachrome conversion activity of dopachrome oxidoreductase (dopachrome conversion factor) are reduced in skin or hair follicle extracts of pheomelanic mice compared with eumelanic mice. In this study, pheomelanic tissues are compared with eumelanic tissues of the same mice and found to be reduced in their DO and DC activities. Implications are discussed.     

Genetic Controls over Activities of Tyrosinase and Dopachrome Conversion Factor in Murine Melanocytes

We evaluated the three catalytic activities of tyrosinase and one activity of dopachrome conversion factor (DCF) in extracts made from skins of 6-day-old yellow and nonyellow mice. At least one of the catalytic activities of tyrosinase and of DCF correlate with the color of pigment being produced in the hair follicles of the mice. We use these data to evaluate existing hypotheses about the mechanism of the interacting genetic controls over melanogenesis.     

Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in regenerating hairbulb melanocytes of lethal yellow (Ay/a), black (a/a), agouti (AwJ/AwJ/) and albino (a/a/c2J/c2J) mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in particulate and soluble fractions of hairbulb melanocytes of lethal yellow (Ay/a C/C), nonagouti black (a/a C/C), and albino (a/a c2J/c2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, Ay/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (AwJ/AwJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and Ay/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and Ay/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in Ay/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.     

Tyrosinase activity in the first coat of agouti and black mice

Tyrosinase activity was compared in the skin and hair bulbs of young black and agouti mice between 4 and 12 days old. Differences in activity were found to be maximal in both the hair and skin at the time of yellow pigment synthesis in agouti mice. Histological examination suggested that the number of dopa-positive melanocytes is similar in the hair bulbs of agouti and black mice. The level of SH-compounds in the hair bulb was examined and found to be elevated in agouti tissue at the time of phaeomelanin formation. It was shown that sulphydryl compounds such as cysteine and glutathione have an inhibitory effect on tyrosinase, and it is possible that the elevated levels of SH-compounds are responsible for a reduction in tyrosinase activity in agouti mice. In agouti hair bulbs, this effect can be reversed in vitro by addition of copper.     

Tyrosinase Activity (TH,DO, PAGE-Defined Isozymes) and Melanin Production in Regenerating Hairbulb Melanocytes of Lethal Yellow (Ay/a), Black (a/a), Agouti (AwJ/AwJ) and Albino (a/a/c2J/c2J) Mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in participate and soluble fractions of hairbulb melanocytes of lethal yellow (A^y/a C/C), nonagouti black (a/a C/C), and albino (a/a c^2J/c^2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, A^y/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (A^wJ/A^wJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and A^y/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and A^y/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in A^y/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Genotype-dependent participation of coat color gene loci in the behavioral traits of laboratory mice

To evaluate if loci responsible for coat color phenotypes contribute to behavioral characteristics, we specified novel gene loci associated with social exploratory behavior and examined the effects of the frequency of each allele at distinct loci on behavioral expression. We used the F2 generation, which arose from the mating of F1 mice obtained by interbreeding DBA/2 and ICR mice. Phenotypic analysis indicated that the agouti and albino loci affect behavioral traits. A genotype-based analysis revealed that novel exploratory activity was suppressed in a manner dependent on the frequency of the dominant wild-type allele at the agouti, but not albino, locus. The allele-dependent suppression was restricted to colored mice and was not seen in albino mice. The present results suggest that the agouti locus contributes to a particular behavioral trait in the presence of a wild-type allele at the albino locus, which encodes a structural gene for tyrosinase.     

The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis

The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis.     

A Transgenic Mouse Assay for Agouti Protein Activity

The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.     

Decreased dopachrome oxidoreductase activity in yellow mice

Dopachrome oxidoreductase (DCOR) is a newly characterized enzyme in the melanin synthetic pathway, active in the conversion of dopachrome to 5,6-dihydroxyindole. DCOR and tyrosinase activity were measured in skin anagen hairbulbs from lethal yellow (Ay/a), sienna yellow (Asy/a) and recessive yellow (e/e) mice with and without treatment with melanocyte-stimulating hormone (MSH). DCOR activity was low (Asy/a) or absent (Ay/a, e/e) in yellow mice without MSH treatment, and increased dramatically in the lethal and sienna yellow mice with MSH. There was no increase in DCOR activity in recessive yellow mice with MSH. Corresponding tyrosinase activity was reduced in lethal yellow and sienna yellow mice without MSH, and increased with MSH. Tyrosinase activity was normal in recessive yellow mice without MSH and did not change with MSH. We conclude that DCOR is an MSH-sensitive enzyme and that DCOR activity is absent in recessive yellow melanocytes. The latter finding suggests that the extension locus may be the DCOR locus.     

The genetics of coat colors in the mongolian gerbil (Meriones unguiculatus)

Genetic studies demonstrated three loci controlling coat colors in the Mongolian gerbil. F1 hybrids of white gerbils with red eyes and agouti gerbils with wild coat color had the agouti coat color. The segregating ratio of agouti and white in the F2 generation was 3:1. In the backcross (BC) generation (white x F1), the ratio of the agouti and white coat colors was 1:1. Next, inheritance of the agouti coat color was investigated. Matings between agouti and non-agouti (black) gerbils produced only agouti gerbils. In the F2 generation, the ratio of agouti to non-agouti (black) was 3:1. There was no distortion in the sex ratios within each coat color in the F1, F2 and BC generations. This indicated that the white coat color of gerbils is governed by an autosomal recessive gene which should be named the c allele of the c (albino) locus controlling pigmentation, and the agouti coat color is controlled by an autosomal dominant gene which might be named the A allele of the A (agouti) locus controlling pigmentation patterns in the hair. The occurrence of the black gerbil demonstrated clearly the existence of the b (brown) locus, and it clearly indicated that the coat colors of gerbils can basically be explained by a, b, and c loci as in mice and rats.     

Tyrosinase activity and the expression of the agouti gene in the mouse

Tyrosinase activity at the time of phaeomelanin synthesis in neonatal mice is lower in agouti than in black skin and hair bulb tissue, and this depressed activity is associated with a reduction in the electrophoretically distinct de novo form of the enzyme. Direct chemical measurements of sulphydryl compounds show elevated levels in agouti hair bulb tissue at this stage of development. The addition of exogenous copper to hair bulb extracts raises the activity of tyrosinase in agouti to approximately the black level but has no affect on black itself. These results are discussed in relation to the role of sulphydryl compounds and copper availability in regulating tyrosinase activity and turnover.     

Ultrastructural change of melanosomes associated with agouti pattern formation in mouse hair.

We have studied the structural alteration of melanosomes in the melanocytes of agouti mice whose genetic characteristic is to produce eumelanin and phaeomelanin alternately in a single hair bulb. Melanocytes of hair bulbs from 1 to 2 day old mice of the black phase were observed to contain rod-shaped melanosomes of the eumelanin type (eumelanosome). In the melanocytes of the hair bulbs from 4 to 6-day old skin, which exclusively contain phaeomelanin, spherical melanosomes (phaeomelanosomes) were seen. On the other hand, the mice of the transitional phase from black to yellow possessed melanocytes that contained both eumelanosomes and phaeomelanosomes within a single cell. This result indicates that the shift from the eumelanin formation to the phaeomelanin formation or vice versa in agouti hair occurs within a single melanocyte.We observed multivesicular bodies in both the agouti melanocytes of the yellow phase and the genotypically yellow melanocytes. These bodies are considered to be the precursor of the phaeomelanin-containing melanosome. They are sometimes observed to have continuity with E. R. suggesting that the melanosomes are derived from E. R. in the phaeomelanin-forming melanocytes.     

Female Fertility and Mating Type Effects on Effective Population Size and Evolution in Filamentous Fungi

The murine agouti locus regulates a switch in pigment synthesis between eumelanin (black/brown pigment) and phaeomelanin (yellow/red pigment) by hair bulb melanocytes. We recently described a spontaneous mutation, hypervariable yellow (A(hvy)) and demonstrated that A(hvy) is responsible for the largest range of phenotypes yet identified at the agouti locus, producing mice that are obese with yellow coats to mice that are of normal weight with black coats. Here, we show that agouti expression is altered both temporally and spatially in A(hvy) mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual A(hvy) mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5' RACE and genomic PCR products revealed that A(hvy) resulted from the integration of an intracisternal A particle (IAP) in an antisense orientation within the 5' untranslated agouti exon 1C. This retrovirus-like element is responsible for deregulating agouti expression in A(hvy) mice; agouti expression is correlated with the methylation state of CpG residues in the IAP long terminal repeat as well as in host genomic DNA. In addition, the data suggest that the variable phenotype of A(hvy) offspring is influenced in part by the phenotype of their A(hvy) female parent.     

In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody

Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.     

Dose-response effects of ectopic agouti protein on iron overload and age-associated aspects of the Avy/a obese mouse phenome

Isogenic and congenic offspring from matings of inbred black a/a dams by sibling (or non-sibling from another inbred strain) yellow agouti Avy/a sires provide an animal model of obese yellow agouti Avy/a and isogenic lean pseudoagouti Avy/a mice exhibiting two different in vivo concentrations (high, very low) of ectopic agouti protein (ASP) with congenic lean black a/a mice as null controls. This makes it possible to differentiate between the high and very low dose levels of ectopic ASP with respect to interactions with diverse physiological and molecular pathways. Assay of differential responses to 12 or 24 months of carbonyl iron overload assessed the possible suitability of this animal model for the study of hemochromatosis. Agouti A/a B6C3F1 mice were used as non-congenic null controls. The age-related waxing and waning of body weight, food consumption, and caloric efficiency, as well as associated changes in pancreatic islets and islet cells, and formation of liver tumors were assayed. While the hypothesis that these mice might serve as a tool for investigating hemochromatosis was not confirmed, the data did provide evidence that even the very low levels of ASP in pseudoagouti Avy/a mice affect the network of molecular/metabolic/physiological response pathways that comprises the yellow agouti obese phenome. We suggest that the combination of yellow agouti Avy/a, pseudoagouti Avy/a, and black a/a congenic mice provides a practical tool for applying a dose-response systems biology approach to understanding the dysregulatory influence of ectopic ASP on the molecular-physiological matrix of the organism.     

Developmental changes of the proliferative response of mouse epidermal melanocytes to skin wounding

A cut was made on the middorsal skin of mice of various ages of strain C57BL/0J using fine iridectomy scissors. Specimens from the wounded skins were fixed at various days after wounding and were subjected to the dopa reaction and to the combined dopa-premelanin reaction. When the dorsal skins of 1.5-day-old mice were wounded, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically in the epidermis adjacent to a skin wound. Pigment-producing melanocytes in mitosis were frequently found in the vicinity of a wound immediately after wounding. When the dorsal skins of 4.5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was smaller than that of 1.5-day-old mice. The increase in number of pigment-producing melanocytes in mitosis was reduced and delayed as compared to 1.5-day-old mice. When the dorsal skins of 8.5-, 20.5-, and 60.5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was much smaller than the newborn mice. Moreover, pigment-producing melanocytes in mitosis were never found. These results indicate that the proliferative response of mouse epidermal melanocytes to skin wounding becomes delayed and diminished with development.     

Developmental interactions in the pigmentary system of the tip of the mouse tail: effects of coat-color genes on the expression of a tail-spotting gene

The tails of agouti C3H/HeJmsHir mice are completely pigmented, whereas the tails of black C57BL/10JHir animals possess unpigmented tips. Genetic analysis indicates that white tail-tipping is due to an autosomal recessive gene, with incomplete penetrance, that segregates independently from the gene for agouti with a maternal influence in the F1 generation. To analyze the influence of specific coat-color genes on the expression of tail-spotting in mice, five congenic lines of C57BL/10JHir with different coat colors were prepared. No influence was observed on the occurrence of tail-spotting in agouti (A/A) or dilute (d/d) mice or in F1 mice from crosses between black and albino (c/c), or in F1 mice from crosses between black and pink-eyed dilution (p/p). However, the frequency of tail-spotting was dramatically decreased in brown (b/b) mice. These results suggest that the mutant allele (b) at the brown locus is involved in determining the extent of pigmented areas in the tail tips of mice through an interaction with the tail-spotting gene.