Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper transcription factor that is important for the development of mast cells. Cultured mast cells (CMCs) of mi/mi genotype express abnormal MITF (mi-MITF), but CMCs of tg/tg genotype do not express any MITFs. It was previously reported that mi/mi CMCs showed more severe abnormalities than tg/tg CMCs, indicating that mi-MITF had inhibitory function. Whereas mi-MITF contains a single amino acid deletion in the basic domain, MITF encoded by mi(ew) allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain. Here the effect of a large deletion of the basic domain was examined. In mi(ew)/mi(ew) CMCs, the expression pattern of genes whose transcription was affected by MITF was comparable to that of tg/tg CMCs rather than to that of mi/mi CMCs. This suggested that ew-MITF lacked any functions. The part of the basic domain deleted in ew-MITF appeared necessary for either transactivation or inhibition of transactivation.

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.

Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mi(ce)/mi(ce) mutant mice of which MITF lacks the zipper domain.

The mi transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amount of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. Mast cells of mi/mi mice show more severe abnormalities than those of tg/tg mice, indicating that the mi-MITF possesses the inhibitory function. The MITF encoded by the mi(ce) mutant allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain using mi(ce)/mi(ce) mice. The amounts of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) messenger RNAs decreased in mast cells of mi(ce)/mi(ce) mice to levels comparable to those of tg/tg mice, and the amounts were intermediate between those of +/+ mice and those of mi/mi mice. Gr B mediates the cytotoxic activity of mast cells, and TPH is a rate-limiting enzyme for the synthesis of serotonin. The cytotoxic activity and serotonin content of mi(ce)/mi(ce) mast cells were comparable to those of tg/tg mast cells and were significantly higher than those of mi/mi mast cells. The phenotype of mi(ce)/mi(ce) mast cells was similar to that of tg/tg mast cells rather than to that of mi/mi mast cells, suggesting that the ce-MITF had no functions. The Zip domain of MITF appeared to be important for the development of mast cells. (Blood. 2001;97:2038-2044)

The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes.

The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.

Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture.

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.

Mutational analysis of the modulation of tyrosinase by tyrosinase-related proteins 1 and 2 in vitro.

The albino (tyrosinase, Tyrc), brown (tyrosinase-related protein 1, Tyrp1b) and slaty (tyrosinase-related protein 2, tyrp2slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS-7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity.

Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus.

Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. Here we report the complete exon/intron structure of the mouse Mitf gene and show it to be similar to the human gene. We also found that the mouse gene is transcriptionally complex and is capable of generating at least 13 different Mitf isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia mutations. Two of the mutations are reported for the first time here (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws), Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data presented here, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal.

Spatially restricted hypopigmentation associated with an Ednrbs-modifying locus on mouse chromosome 10.

We have used the varied expressivity of white spotting (hypopigmentation) observed in intrasubspecific crosses of Ednrb(s) mice (Mayer Ednrb(s)/Ednrb(s) and C3HeB/FeJ Ednrb(s)/Ednrb(s)) to analyze the effects of modifier loci on the patterning of hypopigmentation. We have confirmed that an Ednrb(s) modifier locus is present on mouse Chromosome 10. This locus is now termed k10, using the nomenclature established by Dunn in 1920. The k10(Mayer) allele is a recessive modifier that accounts for almost all of the genetic variance of dorsal hypopigmentation. Using intercross analyses we identified a second allele of this locus or a closely linked gene termed k10(C3H). The k10(C3H) allele is semidominant and is associated with the penetrance and expressivity of a white forelock phenotype similar to that seen in Waardenburg syndrome. Molecular linkage analysis was used to determine that the k10 critical interval was flanked by D10Mit10 and D10Mit162/D10Mit122 and cosegregates with mast cell growth factor (Mgf). Complementation crosses with a Mgf(Sl) allele (a 3-5-cM deletion) confirm the semidominant white forelock feature of the k10(C3H) allele and the dorsal spotting feature of K10(Mayer) allele. MgF was assessed as a candidate gene for k10(Mayer) and k10(C3H) by sequence and genomic analyses. No molecular differences were observed between the Mayer and C57BL/6J alleles of MgF; however, extensive genomic differences were observed between the C3HeB/FeJ and C57BL/6J alleles. This suggests that alteration of MgF expression in C3H mice may account for the k10(C3H) action on white forelock hypopigmentation. Crosses of Ednrb(s) with Kit(WJ-2) (the receptor for MGF)-deficient mice confirmed the hypothesis that synergistic interaction between the Endothelin and MGF signaling pathways regulates proper neural crest-derived melanocyte development in vivo.

The inbred mouse in pigmentation research: significance of a congenic developmental system.

Pigment mutations in inbred mice have been important to many new scientific developments over the past century. Inbred mice are essentially genetically alike because of 10-20 generations or more of sibling mating or the equivalent. Mice of the same inbred strain that differ at only one locus can be used to evaluate the phenotypic effects of that one locus without complication of variation at other loci. Similarly, genic interactions among the functions of two or more loci are evaluated by comparing them in all combinations against a uniform genetic background. The next logical step in describing the pigment system will occur when all pigment cell biologists who use mice (cells, tissues, DNA, RNA) make certain that their mice are congenic with C57BL/6J. As a result, the work of all investigators will be genetically comparable. Their work will also be comparable to those investigating other organ systems, because NIH has chosen C57BL/6J as one of its two standard strains. As a result of this standardization, interactions among the different gene loci that function in the pigment system will become more readily evident and the community of pigment cell biologists using congenic mice will be able to analyze the functional interplay of loci that regulate the entire pigment system in the same way that earlier researchers analyzed one mutant allele, or the interactions of two mutant loci.

Strain-specific white-spotting patterns in laboratory mice.

White spotting is the absence of melanocytes (pigment cells) from part or all of the locations in the body where they are normally found. At least in the case of the W (kit) locus, white spotting has been attributed to apoptosis. In addition to the death of melanoblasts, white spotting might result from their failure to migrate to their normal locations. These developmental failures are known to be melanocyte-specific in some instances and environment-specific in others. The environment is defined as the tissues surrounding the melanoblast. Patterns of white spotting were examined on mice mutant at the piebald (s), patch (Ph), dominant spotting (W(J2)) rumpwhite (Rw) or belted (bt) loci. The dominant spotting locus has been cloned and found to encode KIT; it has been suggested that Patch encodes the linked alpha-PDGF receptor. Piebald encodes the endothelin beta receptor. In each case, the phenotypes expressed when the allele was backcrossed onto one inbred strain C57BL/6 (B6), were compared with phenotypes expressed when the allele was backcrossed onto a different inbred strain, JU/CtLm (JU). The literature documents genetic loci that influence the extent of the white spotted area; we herein demonstrate that genetic loci also influence the location where the white spot (absence of melanocytes) will occur over the body of the mouse. Spotting occurs in a more anterior direction on JU mice that are piebald, patch or dominant-spotted compared with similar B6 mice. The relationship is reversed in rumpwhite mice, where white spotting is more anterior in the C57BL/6 mice than in the JU mice. The spotting pattern of belted mice was not modified by the background genome. Thus, the Mendelian observations indicate that several loci, which differ in JU compared with B6 mice, influence the size and the location of white spots on the mouse.

Mahogany (mg) stimulates feeding and increases basal metabolic rate independent of its suppression of agouti.

The mahogany (mg) locus originally was identified as a recessive suppressor of agouti, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of agouti cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that mg, when homozygous, blocks not only the ability of agouti to induce a yellow coat color when expressed in the skin of the lethal yellow mouse (AY), but also the obesity resulting from ectopic expression of agouti in the brain. Detailed analysis of mg/mg AY/a animals, presented here, demonstrates that mg/mg blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however, mg/mg did not reduce hyperphagia in the AY/a mouse. Furthermore, mg/mg induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of AY. Consequently, although mahogany is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of agouti.

Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo.

Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA-Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of alpha-MSH, but its action cannot be explained solely by inhibition of alpha-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by alpha-MSH, or by Agrp, which indicates that alpha-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.

Genetic studies of the mouse mutations mahogany and mahoganoid.

The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1re) or a gain-of-function Agouti mutation (lethal yellow, Ay). We found that the Mc1re mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of Ay on both coat color and obesity. Plasma levels of alpha-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.

Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin.

In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

In vivo and in vitro morphological analysis of melanocytes homozygous for the misp allele at the murine microphthalmia locus.

The misp allele (microphthalmia-spotted), a mutant allele at the murine microphthalmia (mi) locus, when homozygous, results in a normal phenotype in which there is no apparent alteration in pelage pigmentation or ocular development. However, when heterozygous with other mi locus alleles, specifically Miwh (microphthalmia-white) the misp allele exerts an affect on the phenotype. We examined the ultrastructure of melanocytes in the anagen hair bulb and the choroid plus the retinal pigmented epithelium of C57BL/6J-misp/misp mice, C57BL/6J-Miwh/Miwh mice, C57BL/6J-Miwh/misp mice, and C57BL/6J-(+)/+ control mice. Melanocytes of the misp/misp mice appeared normal in situ. However, melanocyte cultures derived from neonatal skins of misp/misp mice exhibited small primary colonies that did not dramatically expand in size. Occasionally, abnormalities in the structure of the Golgi apparatus were observed in primary cultures of misp/misp melanocytes. These results demonstrate that while the misp allele has no obvious effect on the phenotype of the mouse, it does dramatically suppress the survival of melanocytes in normal culture conditions.

Misrouting of tyrosinase with a truncated cytoplasmic tail as a result of the murine platinum (cp) mutation.

Mice homozygous for the platinum (cp) allele at the albino locus manifest severe oculocutaneous albinism despite the presence in vitro of tyrosinase activity at 25% wild-type levels. We demonstrate that the cp allele results from an A-->T substitution, changing a lysine residue at position 489 to a termination codon, with truncation of tyrosinase's cytoplasmic tail. In choroidal melanocytes of neonatal mutant mice, tyrosinase activity could be detected in the trans Golgi network, but was absent from melanosomes. Instead, it was detected in vesicles in the cell periphery and dendrites, and on the extracellular surface. In the retinal pigment epithelium, activity was present on the extracellular apical and basolateral surfaces. Our results demonstrate misrouting of a mutant tyrosinase lacking its cytoplasmic tail, providing an explanation for the severe effect of this mutation on ocular and cutaneous pigmentation.

The pinkeyed-dilution protein and the eumelanin/pheomelanin switch: in support of a unifying hypothesis.

The two major types of mammalian melanin are pheomelanin (yellow or red pigment) and eumelanin (black or brown). The agouti (A) and extension (E) loci determine whether follicular melanocytes will deposit pheomelanin or eumelanin within their melanosomes. Mutations at the murine pinkeyed-dilution (P) locus cause a striking reduction in deposition of eumelanic, but not pheomelanic, pigment. The mRNA encoded at the P locus is not expressed in skin that exclusively produces pheomelanic pigment as a result of mutation at the agouti locus. We have suggested, based upon both genetic and biochemical evidence, that three key melanogenic proteins--tyrosinase, tyrosinase-related-protein-1 (TRP-1), and TRP-2, encoded at the albino (C), brown (B), and slaty (Slt) loci, respectively--form a high-molecular-weight "melanogenic complex" within the melanosome. High-molecular-weight forms of tyrosinase, TRP-1 and TRP-2, are absent from eumelanic ocular tissues of p(un)/p(un) mice that fail to produce normal P-locus transcript, even though these mice are genetically normal at the loci that regulate production of the three melanogenic proteins. We have hypothesized that the presence of the p-locus protein is important for the integrity of the melanogenic complex and for the levels of members of the TRP family. We show here that the yellow skins of mice mutant at the agouti or extension loci, as well as the nonyellow skins of pinkeyed-unstable (p(un)/p(un)) mice, demonstrate greatly diminished levels of tyrosinase, TRP-1 and TRP-2, and an absence or markedly decreased proportion of high-molecular-weight forms of melanogenic proteins. We conclude that normal levels of wild-type P-locus protein are necessary for eumelanogenesis and that the absence of this protein may be necessary, but is not sufficient to cause the melanosome to switch to the production of pheomelanin. We discuss the implications of our results in relation to the interacting genetic controls regulating melanogenesis.

Comparative analysis of melanins and melanosomes produced by various coat color mutants.

Pigmentation in the skin, hair, and eyes of animals is influenced by a number of genes that modulate the activity of melanocytes, the intervention of enzymatic controls at different stages of the melanogenic process, and the physico-chemical properties of the final pigment. The results of combined phenotypic, ultrastructural, biochemical, and chemical analyses of hairs of a variety of defined genotypes on a common genetic background performed in this study are consistent with the view that pigmentation of dark to black hairs results from the incorporation of eumelanin pigments whereas that of yellow hairs results from the incorporation of eu- and pheomelanins. It is also clear that relatively minor differences in melanin content can have dramatic effects on visible hair color. A good correlation was found for expression of (and enzyme activities associated with) TRP1 and TRP2 with eumelanin synthesis and eumelanosome production.

Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.