Mouse coat color mutations: From fancy mice to functional genomics

Authors


Abstract

Mouse coat color mutations have a long history in biomedical research. The viable and visible phenotype of most coat color mutations has made the pigment cell, the melanocyte, an ideal system for genetic, molecular, and cellular analysis. Molecular cloning and analysis of many of the different coat color mutations have revealed the roles of a diverse range of genes, and today we know more about the pathways and proteins that regulate the development and function of pigment cells than we know about most other cell types in mammalian organisms. Coat color mutations have also provided novel insights into stem cell biology and human diseases, including melanoma. In the future, it will be important to build on this history and knowledge by taking advantage of the extensive repertoire of recently developed genome-wide methodologies, available genomic information, and the powerful methods that have been developed for modifying the mouse genome to systematically dissect the development and function of this important cell type. The usefulness of coat color mutations has just begun to emerge. Developmental Dynamics 235:2401–2411, 2006. © 2006 Wiley-Liss, Inc.

INTRODUCTION

The mouse is the mammalian model system of choice for experimental genetic analysis. A major reason for this choice are the many coat color mutations in the mouse, now totaling approximately 130 different genes with nearly 1,000 different alleles, many of which have been available for centuries. In addition to providing strikingly colored mice for “mouse fanciers,” it was realized during the early part of the 19th century that the coat color mutations provided a visible tool for genetic analysis. One of the first uses of mouse coat color mutations in genetics was to prove that Mendel's laws operated in mammals (reviewed by Silver,1995). A few years later, coat color mutations were used during the generation of the first inbred mouse strain, DBA (named according to the visible coat color mutations dilute (d), brown (b), and agouti (a) that it carries). This finding was a major development for the experimental use of mice. Coat color mutations are still important as visible markers today, for example, as visible markers in general genetic analysis, as markers for the many different inbred lines developed since DBA, during the generation of mouse knockout mutations, and for the generation of mouse balancer chromosomes (Zheng et al.,1999; Hentges and Justice,2004). Most importantly, however, coat color mutations have provided an opportunity to study the development and biology of an important, vertebrate-specific cell type, the melanocyte. In addition, these mutations have provided models for several different human diseases.

Melanocytes are the cells responsible for pigment synthesis in skin and hair of vertebrates. Although melanocytes also populate several other regions of the body, including the inner ear where they are essential for normal hearing, the choroid and iris of the eye, and the Harderian gland, we will limit our discussion to the melanocytes of the skin and hair. In mice, the precursors to melanocytes, the unpigmented melanoblasts, arise from the neural crest at approximately 8.5 days in embryonic development (E8.5) (Fig. 1A). They reside transiently in the “migration staging area,” a region demarcated by the neural tube, the somite, and the overlying epithelium, and then migrate along a dorsolateral pathway between the dermatome and the overlying ectoderm, then ventrally through the developing dermis. At 12.5–13.5 days of development they move into the overlying epidermis and eventually, into the developing hair follicles (Mayer,1973; Wehrle-Haller and Weston,1995). Once in the hair follicle, the melanoblasts are separated into two populations: differentiated melanocytes in the hair matrix, which start pigment synthesis approximately 4 days after birth, and melanocyte stem cells, which reside in the so-called bulge region of the hair follicle and generate new melanocytes during each hair cycle (Nishimura et al.,2002; Fig. 1B). Mak et al. (2006) have shown recently that the first melanocytes that produce pigment in the hair originate from melanoblasts which bypass the melanocyte stem cell stage.

Figure 1.

Melanocyte development. A: Melanoblasts, the precursors to melanocytes, arise from the neural crest and then migrate to their destination, e.g., in the hair follicle. Several genes known to affect the development of melanoblasts and melanocytes are indicated. B: When in the hair follicle, the melanoblasts either go straight to the hair follicle matrix and form fully developed melanocytes or they go to the bulge region where they form melanocyte stem cells. During each hair cycle, the melanocyte stem cells renew themselves and generate novel melanocytes. Red represents proposed amplifying cells and green melanocytes.

The interesting life cycle of melanocytes makes them a particularly attractive model for studying the molecular basis of cellular function, including how a particular cell type arises, how its proliferation and survival is regulated, how it travels to its destination, and how it differentiates. Much of what we know about melanocytes has been gained through research on the mouse coat color mutations, which in addition to being readily visible in living animals, are often nonlethal and, therefore, easy to maintain and breed. These mutations affect various different processes during the melanocyte lifecycle, including their initial development and maintenance, their migration, as well as the formation and processing of pigment. More than half of the coat color mutations have been molecularly cloned and characterized, and, together with the phenotypic information, the molecular and biochemical analysis has resulted in an unusual depth of understanding of the function of a single cell type. The cloning of the remaining loci will undoubtedly lead to important insights as well.

The nature and effects of coat color loci have been reviewed recently (Bennett and Lamoreux,2003; Tomita and Suzuki,2004), and a public Web site maintains information on all known coat color mutations and genes in the mouse, both those that have been cloned and those that have not (Oetting and Bennett,2006http://ifpcs.med.umn.edu/micemut.htm). Broadly speaking, the coat color mutations have been classified into several different categories according to their effects on the melanocyte. Here, we will briefly discuss each category of mutations and recent developments as well as prospects for the future.

Development of the Melanocyte

The first and perhaps most interesting category of coat color mutations affect melanoblast/melanocyte development due to effects on differentiation, migration, survival, and/or proliferation. The best studied of these mutations are dominant white spotting (W, now Kit) and steel (Sl, now Kitl), affecting the loci that encode the receptor-ligand pair Kit (a receptor tyrosine kinase) and Kit-ligand (Kitl, also known as stem cell factor, mast cell growth factor, and steel factor), respectively. Severe mutations at the Kit and Kitl loci result in black-eyed white mice with hematopoietic and germ cell defects, showing that these genes are necessary for the development of melanocytes, germ cells, and hematopoietic stem cells, but not for retinal pigment epithelial cells of the eye. Melanoblasts in mice homozygous for loss-of-function mutations in either gene remain in the migration staging area and then disappear when traced by the expression of the melanocyte-specific gene Dct (see below; Wehrle-Haller and Weston,1995). This finding suggests that these genes are necessary for melanoblast survival and/or for their dispersal from the migration staging area. Jordan and Jackson (2000) have shown that, whereas Kitl acts as a chemokinetic factor that accelerates melanoblast movement to the hair follicles, it does not have chemoattractive abilities.

Mice carrying mutations in the microphthalmia-associated transcription factor gene Mitf have overlapping phenotypes with Kit and Kitl mice in that all of these mutations affect melanocytes and mast cells (reviewed in Steingrimsson et al.,2004). Mitf mutant mice, however, have normal germ cell development. Some of the Mitf mutations also result in severe microphthalmia and some cause osteopetrosis, whereas Kit and Kitl mice are normal with respect to eye and bone development. The Mitf gene encodes a bHLHZip transcription factor that has been shown to regulate the expression of many melanocyte-specific genes (reviewed in Steingrimsson et al.,2004). In Mitf mutant embryos, a few melanoblasts initially arise from the neural crest but then they disappear shortly thereafter. Experimental evidence suggests that Mitf is important for promoting the transition of precursor cells to melanoblasts and essential to secure melanoblast survival (Opdecamp et al.,1997; Hornyak,2001). Consistent with this finding, Mitf has been shown to regulate the expression of the antiapoptotic gene Bcl2 (McGill et al.,2002), which is essential for the survival of both melanoblasts and melanocytes (Mak et al.,2006). In addition, multiple lines of evidence suggest that Mitf can act as a determinant of melanocyte development; ectopically induced expression of Mitf in various cell lines and organisms results in the production of cells with pigment cell characteristics (Tachibana et al.,1996; Lister et al.,1999; Planque et al.,1999; Bejar et al.,2003). Thus, Mitf is considered to be a master regulator of melanocyte development. Although considerable knowledge is available on how the expression of Mitf is regulated and although several Mitf target genes are known (reviewed in Steingrimsson et al.,2004), little is currently known about the Mitf target genes that mediate melanoblast and melanocyte development.

The similar melanocyte phenotypes of Kit, Kitl, and Mitf mutant mice suggest that these genes represent components of a signal transduction pathway where either Kit signals to Mitf, or Mitf regulates expression of Kit. There is experimental evidence supporting both scenarios. It is well established in vitro that Kit signals to Mitf through Map kinase-mediated phosphorylation of Mitf (reviewed in Steingrimsson et al.,2004). This pathway promotes the interaction of Mitf with the coactivator p300, resulting in increased transcriptional activation potential of the Mitf protein. The same signaling pathway also results in decreased stability of Mitf through proteasome-mediated degradation. Thus, Kit signaling leads to a more active but less stable Mitf protein. It has also been shown that Mitf can activate Kit expression in mast cells (Tsujimura et al.,1996) and in melanoblasts. Although Mitf up-regulates Kit expression, the onset of Kit expression does not depend on Mitf (Opdecamp et al.,1997). Clearly, as melanocytes form from the neural crest, rest in the migration staging area, and then travel to their destinations, they enter and sense different environments at each stage. It is entirely possible, therefore, that each of the two scenarios described above apply at different stages of melanocyte development and that at one stage Mitf up-regulates Kit expression and at another, Kit signaling to Mitf leads to direct effects on transcription. Perhaps this forms a positive feedback mechanism to stabilize the development of this cell type.

Several other interesting coat color mutations also affect transcription factor genes and receptor-ligand pairs. The endothelin3 (Edn3)/endothelin receptor B (Ednrb) ligand-receptor pair is encoded by the lethal spotting (ls) and piebald spotting (s) coat color genes, respectively. Ednrb is a seven-transmembrane G-protein coupled receptor that responds to the Edn3 and Edn1 ligands (reviewed in McCallion and Chakravarti,2001). Mice that carry a deletion of the Ednrb locus are almost completely white and develop megacolon (Hosoda et al.,1994). Similarly, mice that carry a mutation at Edn3 have white spots and suffer from agangliosis, resulting in megacolon (Baynash et al.,1994). At 10.5 days of development, Ednrb homozygous mutant embryos have fewer melanoblasts than wild-type embryos (Hosoda et al.,1994). By inducing the expression of Ednrb at different times of embryonic development, Shin et al. (1999) showed that Ednrb signaling is important for the initiation and correct migration of melanoblasts between E10 and E12.5. Of interest, Ednrb signaling is not required for the initial specification of melanoblasts.

Like Ednrb and Edn3 mutations, mutations in the Sox10 transcription factor gene also affect melanocytes and enteric neurons. Heterozygotes carrying a loss-of-function mutation in Sox10 have white head and belly spots, and enteric agangliosis, whereas in homozygotes, melanoblasts, and peripheral glia are missing. Homozygous Sox10 mice die between embryonic days 16.5 and 18.5 of development (Britsch et al.,2001). The similarity in phenotypes of Sox10, Ednrb, and Edn3 mutant mice indicated that this finding represented a direct hierarchical pathway. However, recent data suggest this is not the case and that the Edn3/Ednrb and Sox10 pathways are distinct and not hierarchical, at least in melanocytes (Hakami et al.,2006). On the other hand, a direct hierarchical relationship has been shown between Sox10 and Mitf. The Sox10 protein binds sequences in the Mitf promoter and activates expression of Mitf in melanocytes (reviewed in Mollaaghababa and Pavan,2003). Some studies suggest that this Sox10-mediated activation of Mitf also requires Pax3, the transcription factor encoded by the Splotch coat color locus, whereas other studies have failed to detect such synergistic interactions (reviewed in Steingrimsson et al.,2004). Interestingly, mutations in the human PAX3, MITF, EDN3, EDNRB, and SOX10 genes are associated with different types of Waardenburg syndrome (WS). PAX3 mutations are associated with most, if not all, cases of WS type 1, MITF mutations with some forms of WS type 2a, and WS type 4 (also known as Waardenburg-Shah and Hirschsprung disease Type II) is associated with mutations in the EDN3, EDNRB, or SOX10 genes. These genes, therefore, are all involved in melanocyte development in humans, just as in mice. Recently, mutations in the SNAI2 gene, encoding a zinc finger transcription factor, have been reported in individuals with WS type 2D (Sanchez-Martin et al.,2002), and this gene has emerged as an important marker for early melanoblast development. In mice, knockout mutations in Snai2 result in mice with coat color dilution and white spots, suggesting that the function of the gene is conserved between mice and humans (Perez-Losada et al.,2002).

Melanocyte Stem Cells

In addition to playing a role in melanoblast/melanocyte development, recent evidence has shown the importance of Mitf, Pax3, and Sox10 in the maintenance of melanocyte stem cells in the hair follicle. Melanocyte stem cells (MSCs) have emerged as a model for stem cell research due to their anatomical separation from their more differentiated progeny. They are also believed to present a unique opportunity for studying the interactions between stem cells and their niche. We recently have reviewed the role of the above-mentioned genes in the function and maintenance of MSCs (Steingrimsson et al.,2005). The work of Nishimura and colleagues (2005) has suggested that Mitf is expressed in MSCs where it is necessary for regulating the expression of the Bcl2 gene so as to guarantee survival. Recently, the role of Bcl2 in MSCs has been analyzed carefully by Mak et al. (2006) and associates who showed that Bcl2 is essential for the emergence of MSCs. However, in contrast to Nishimura and colleagues, who suggested that MSCs are initially present in the bulge region of Bcl2 mutant mice and then disappear at 6.5 days after birth, Mak et al. do not detect any MSCs in the bulge region of hair follicles from Bcl2 mutant embryos. This finding suggests that the Bcl2 protein is required early for melanoblast survival and may not be important for the MSCs. Of interest, Bouillet et al. (2001) have shown that all the effects of Bcl2 are relieved in animals simultaneously mutant for the gene encoding the BH3-only protein Bim, suggesting that all the effects of Bcl2 are due to the action of Bim.

Pax3 is also expressed in MSCs where it supposedly activates the expression of Mitf, resulting in its accumulation in the MSC (Lang et al.,2005). At the same time, Pax3 represses the Mitf/Sox10-mediated activation of Dct, an MSC marker gene. Activated β-catenin relieves the Pax3-mediated repression of Dct, resulting in immediate and rapid expression. According to this model, Pax3 and Wnt signaling regulate differentiation as the MSCs divide to regenerate themselves and to produce more differentiated progeny. Recently, Osawa et al. (2005) characterized in detail the expression of several melanocyte-specific genes in the MSCs, using both immunohistochemistry as well as a single-cell transcript analysis methodology. They were able to show that expression of the Sox10, Kit, and Mitf genes was undetectable or reduced in MSCs, whereas Pax3 and Dct were both expressed at high levels. All these genes were expressed in differentiated melanocytes. This finding suggests that melanoblasts, which enter the niche in the bulge region of the hair follicle, turn off expression of these genes, perhaps as they become quiescent. The low level of Mitf and Sox10 gene expression in the MSCs does not support the model of Lang et al. (2005), whereas the high level of Pax3 expression is consistent with the importance of this transcription factor in the nodal point of MSC differentiation.

Melanosome Formation

Another important and large class of coat color mutations affect the formation of cellular organelles, including melanosomes, the melanocyte-specific organelles where pigment is stored and synthesized. There are two major types of melanosomes: eumelanosomes, which contain black and brown eumelanin; and pheomelanosomes, which contain red to yellow pheomelanin. The melanosomes form as round vesicles that bud from the Golgi–endoplasmic reticulum–lysosome complex (GERL); an alternative view is that they form from endosomes (Theos et al.,2005). Both may be true in that a primary melanosome buds from GERL, whereas some melanosomal components are routed by means of endosomes. The initial stage I melanosomes transform into stage II melanosomes, elongated fibrillar organelles that express the pigmentation enzyme tyrosinase (Fig. 2A). The synthesis and deposition of the pigment melanin, a complex polymer of indolequinone and dihydroxyindole carboxylic acid, takes place in stage III melanosomes. If the melanosome becomes electron-dense with melanin, stage IV melanosomes have formed. At least 20 coat color mutations are known to affect the formation of the melanosome. These mutant mice are hypopigmented, many have increased bleeding times, and some show additional systemic symptoms, often of the kidney or lung. Most of the genes involved have been cloned and shown to encode components of multiprotein complexes, which are necessary for the assembly of several different organelles, including the melanosome. The role of these genes in melanosome assembly has been reviewed recently (Shiflett et al.,2002; Li et al.,2004; Wei,2006) and will not be discussed here except to say that these mutations are models for the human Hermansky–Pudlak and Chediak–Higashi syndromes. These are pleiotropic syndromes whose effects can be traced to defects in the genesis or function of several different organelles, including lysosomes, melanosomes, and platelet dense granules. Thus, the mouse coat color mutations have not only revealed the molecular processes involved in organelle assembly, they also serve as important models for these human syndromes.

Figure 2.

Melanosome development. A: The four stages of melanosome development are shown with the striated stage II melanosome, the early deposition of melanin in stage III and the fully pigmented stage IV melanosomes. B: In stage III melanosomes, the pigmentation enzymes Tyr, Tyrp1, and Dct reside in the melanosome membrane and catalyze the synthesis of melanin from tyrosine. The melanin is believed to be deposited onto fibrillar structures formed by the Silver protein. Currently, it is not known how tyrosine is transported into the melanosome. Several other proteins reside in the melanosome membrane, including the Mart1 and Slc24A5 proteins. Their function is largely unknown.

Melanosome Function

Another class of coat color mutations have uncovered genes that encode for proteins located in the melanosome, primarily the eumelanosome, where they function as structural components, as membrane proteins of unknown function or as pigment synthesizing enzymes. One of the most interesting is Silver (also known as pmel17), the product of the silver (Si) locus. This protein is expressed in stage I melanosomes and is an important component of the fibrillar structures seen in stage II eumelanosomes (Fig. 2B; these structures are lacking in pheomelanosomes) and may be sufficient for their formation (reviewed in Theos et al.,2005). These fibrils are believed to form the basis on which melanin is deposited in stage III melanosomes.

The enzymes that catalyze melanin formation are encoded by the albino (c, tyrosinase, Tyr), slaty (slt, dopachrome tautomerase, Dct), and brown (b, tyrosinase-related-protein1, Tyrp1) coat color genes. These are transmembrane proteins, which are located in the melanosome membrane (Fig. 2B). The major parts of these proteins are located in the melanosome lumen where they catalyze the synthesis of melanin from the amino acid tyrosine. The function and properties of tyrosinase were reviewed in detail recently (Wang and Hebert,2006) and will not be discussed here except to say that its function is affected by pH. The optimal pH of mammalian tyrosinase is around pH 7, and no activity is observed below pH 5. The internal pH of the melanosome changes from acidic to neutral as the melanosome develops from stage II to stage III (Raposo et al.,2001), suggesting that pigment synthesis may be regulated by internal pH. Ancans and associates (Ancans et al.,2001) have suggested that internal pH is regulated by the P protein, a product of the pink-eyed dilution locus. The P protein is a membrane protein with similarity to the Escherichia coli Na+/H+ anti-porter protein. The exact function of the P protein is enigmatic, and the idea that it regulates melanosomal pH is inconsistent with the results of Toyofuku et al. (2002) and Chen et al. (2002), who showed that P protein is involved in processing and sorting of tyrosinase, which is retained in the endoplasmic reticulum in mutant melanocytes. It is also inconsistent with the observation of Staleva et al. (2002), who showed that the P protein may be involved in glutathione metabolism. Regardless of the exact role of the P protein in melanocytes, it is clear that the melanosome membrane plays an important role in melanosome biology. In addition to regulating internal pH, the membrane is likely to also regulate the importation of tyrosine, the initial substrate of melanogenesis; however, nothing is known currently about how tyrosine is imported into the melanosome.

The Slc45a2 protein (also known as Matp) is a transmembrane protein encoded by the underwhite coat color locus (Newton et al.,2001). The P and Slc45a2 proteins are similar in that both have 12 transmembrane regions and, therefore, are likely to function as transporters. Recent studies have shown that processing of tyrosinase and its trafficking to the melanosome is disrupted in Slc45a2 mutant melanocytes, suggesting that Slc45a2 is involved in melanosome formation or maturation (Costin et al.,2003). However, neither the P nor Slc45a2 proteins seem to be located in the melanosome membrane, suggesting that they function in the sorting pathway before melanosomes mature (Costin et al.,2003). Several other proteins, including membrane proteins, are known to be located in the melanosome, including Mlana (also known as Mart1) and Slc24a5 (Fig. 2B). Mlana is a type III membrane protein known to be expressed in stage I and II melanosomes where it seems to be necessary for the proper function of Silver (Hoashi et al.,2005). Unfortunately, no mouse Mlana mutations have been identified, so we do not know the role this protein plays in the melanocyte. Recently, Lamason et al. (2005) reported the isolation of the gene encoded at the golden locus in zebrafish and showed that it encodes a putative cation exchanger called Slc24a5. They also showed that this protein is located in internal membrane-bound structures, most likely the melanosome. Remarkably, they also identified a polymorphism in the human SLC24A5 gene, which is tightly associated with lighter skin pigmentation in humans. This polymorphism, a coding SNP that changes alanine 111 to threonine, is found almost exclusively in people of European descent, whereas the ancestral genotype is found in African, indigenous Americans, and East Asian populations. Lamason and colleagues' studies suggest that this polymorphism explains between 25 and 38% of the difference in skin color of Europeans and Africans. Other vertebrate species, including the mouse, have alanine at this position. The Slc24a5 gene is located on chromosome 2 in the mouse; however, no coat color mutations are known in this region, so we will need to wait for the generation of knockout and knockin mice to analyze its effect on skin and coat color in mice. In humans, mutations in the TYR, TYRP1, P, and SLC45A2 proteins result in Oculocutaneous Albinism of type 1 (OCA1), type 3 (OCA3), type 2 (OCA2), and type 4 (OCA4), respectively. These mutations affect hair, skin, and eye color to various degrees. Currently, no mutations are known in the human DCT or SILV genes.

Several other interesting coat color mutations believed to affect the melanosome, including brownoid, dark, golden, and rimy, are yet to be molecularly cloned and characterized. Their cloning will undoubtedly reveal novel melanosomal components and provide further insights into the function of this interesting organelle.

Melanosome Transport

Mouse coat color mutations have also illuminated our understanding of how melanosomes are transported from the perinuclear region of the melanocyte to the dendrite tips, where they are exported to adjacent keratinocytes. Although little is currently known about how the melanosomes are transferred to keratinocytes, a substantial body of knowledge is available on their intracellular transfer, and the melanosome has emerged as an important model for studying organelle motility (reviewed in Barral and Seabra,2004). The melanosomes in melanocytes of mice homozygous for the dilute (d), ashen (ash), and leaden (ln) coat color mutations accumulate in the perinuclear region of the cell, resulting in a decrease in visible hair pigmentation. Although melanosomes are transported along both actin-based and microtubule-based motors in wild-type melanocytes, these mutations affect the actin-based movements only. The bidirectional microtubule-based movements between the center of the cell and its periphery still take place in mutant melanocytes, but a failure in melanosome capture at the periphery results in their accumulation at the center (Wu et al.,1998). The d, ash, and ln loci encode for the Myosin Va, Rab27a and Melanophilin proteins, respectively (Mercer et al.,1991; Wilson et al.,2000; Matesic et al.,2001). Myosin Va is an actin-based motor protein that contains an N-terminal motor domain that binds to actin and generates force by hydrolyzing ATP, and a C-terminal domain that interacts with the cargo (Fig. 3). Myosin Va has several alternatively spliced exons; exon F is specific to melanocytes (Seperack et al.,1995). Rab27a belongs to the Rab family of Ras-like GTPases, proteins implicated in most steps of vesicular trafficking in the cell. In melanocytes, Rab27a associates with the melanosome membrane and is most concentrated in melanosome-rich dendritic tips (Wu et al.,2001). Melanophilin (also known as Slac2) is a synaptotagmin-like protein that binds both the Rab27a and Myosin Va proteins (Strom et al.,2002; Wu et al.,2002). The N-terminus of melanophilin interacts with Rab27a and the central region with the region encoded by exon F of the globular tail of MyosinVa. Thus, melanophilin provides a bridge between the melanosome-associated Rab27a and the actin-bound Myosin Va, resulting in the capture of melanosomes in the cell's periphery. The melanosomes are then transported to the dendritic extensions where they are transferred to keratinocytes. This transfer of melanosomes is not affected in d mutant mice, suggesting that the Myosin Va pathway is important to ensure that melanosomes are present in the dendritic processes for even transfer to keratinocytes in the hair (O'Sullivan et al.,2004).

Figure 3.

Melanosome capture and transport is regulated by proteins encoded by coat color genes. The Rab27a protein interacts with the melanosome, whereas the Melanophilin protein forms a bridge between Rab27a and Myosin Va. The region encoded by exon F of Myosin Va interacts with melanophilin. The Myosin Va protein is then responsible for capturing the melanosome before its transfer to the dendritic tips.

The mouse dilute suppressor (dsu) mutation reverses the dilute coat color effects seen in d, ash, and ln mutant mice (Sweet,1983; Moore et al.,1988); suppression is more complete for d than ash or ln, and suppression is even seen in dilute loss-of-function mutations. The dsu mutation does not suppress other mutations that dilute mouse coat color (Moore et al.,1990), suggesting that the action of this gene is specific to the Myosin Va pathway. The dsu gene recently has been cloned and shown to encode a 214 amino acid, vertebrate-specific protein of unknown function (O'Sullivan et al.,2004). The gene symbol is now Wdt2, and the allele is written as Wdt2dsu. In Wdt2dsu mutant animals, a large portion of the gene is deleted, suggesting that loss-of-function mutations at this locus can suppress the effects of mutations in the Myosin Va motor. How a loss-of-function mutation in Wdt2dsu can suppress loss-of-function mutations in the MyosinVa gene is not understood currently. O'Sullivan et al., (2004) have proposed that the normal Wdt2 protein functions in the phagocytic process that leads to transfer of melanosomes from melanocytes to keratinocytes or in the way pigment is incorporated into the developing hair. Future studies on the cellular location and action of the Wdt2 protein are needed to resolve these issues.

In humans, mutations in the MYO5A, RAB27A, and MELANOPHILIN genes are associated with Griscelli syndromes 1 (GS1), 2 (GS2), and 3 (GS3), respectively (reviewed by Tomita and Suzuki,2004). Griscelli syndromes are rare disorders characterized by pigment dilution of the skin and hair and accumulation of end-stage melanosomes in the center of the melanocytes. GS1 (also known as Elajalde syndrome) is also associated with neurological impairment and GS2 with an immune defect. Thus, again the mouse mutations have provided models for human diseases and insights into the behavior of an important cellular process.

Regulation of Pigment Type

An interesting group of mouse coat color mutations encode proteins that regulate which type of pigment, eumelanin or pheomelanin, is synthesized in the cell at a given time. The wild-type mouse has agouti coat color; the hair is banded where the tip has black eumelanin, the center has pheomelanin and the base again eumelanin. These bands of pigment are generated by pigment type switching, which is regulated primarily by the melanocortin-1 receptor (Mc1r) and its ligands, α-melanocyte-stimulating hormone (α-MSH) and the agouti signal protein (ASP; reviewed by Schaffer and Bolognia,2001, Wolff,2003; Garcia-Borron et al.,2005). These proteins are encoded by the extension (e, now Mc1r, also known as recessive yellow), pro-opiomelanocortin-1 (Pomc1), and agouti (a) coat color loci, respectively. The e and a loci are classic coat color mutations, whereas the Pomc1 gene has been knocked out by gene targeting, resulting in brownish mice with reduced eumelanin, in addition to other defects (Yaswen et al.,1999). The Pomc1 locus encodes a large precursor peptide that is cleaved into adrenocorticotrophic hormone (ACTH) and α-MSH as well as other peptide hormones not to be discussed here. Thus, the pleiotropic phenotype of Pomc1 mutant mice can be explained by the different activities of the peptides encoded by the gene. The Mc1r protein, a G-protein coupled receptor, is expressed in melanocytes, and upon activation by α-MSH, stimulates the cAMP pathway resulting in melanogenesis and production of eumelanin. The ASP protein inhibits the α-MSH–mediated stimulation of Mc1r, promoting pheomelanin production and yellow coat color. The yellow band in the wild-type mouse hair results from the transient expression of ASP in dermal papilla cells of the hair follicle. There are approximately 100 different alleles available at the a locus in mice with different effects on the phenotype. Mice that carry loss-of-function mutations at the a locus are black, whereas dominant mutations (e.g., due to ectopic expression of ASP) are yellow, as are mice that carry loss-of-function mutations in the Mc1r gene (the e locus). That Pomc1 knockout mice are brownish rather than yellow suggests that the Mc1r receptor can function without the α-MSH ligand or that Mc1r has another melanocyte-specific ligand yet to be isolated. In fact, mice that carry the Pomc1 mutation on a mixed 129;C57BL/6J background (the latter inbred strain is black due to the presence of the a mutation) produce abundant eumelanin, despite the absence of α-MSH (Slominski et al.,2005). Interestingly, human hair is not banded, and the ASP protein does not appear to play a role in human pigmentation, although involvement in skin pigmentation of Africans has been proposed recently (Bonilla et al.,2005). The MC1R receptor, however, does play a role in human pigmentation, because mutations at the locus result in red hair and fair skin in humans (reviewed in Makova and Norton,2005). Similarly, humans carrying a mutation at the POMC locus have red hair, suggesting involvement of this gene in human hair pigmentation (Krude et al.,1998). Because these individuals also lack ACTH, they show other more severe defects. Thus, the role of the α-MSH ligand in determining human hair color is not unequivocal at this point.

Several other coat color mutations are known to affect pigment type switching, including the mahogany, mahoganoid, dorsal-dark stripe, and umbrous mutations. The best understood are the mahogany and mahoganoid mutations, the loci that encode the Attractin (Atrn) and Mahogunin (Mgrn1) proteins, respectively. Both mutations result in a similar darkened phenotype, nearly identical to loss-of-function mutations at the agouti locus. Genetic analysis has shown that these two genes act downstream of agouti but upstream of Mc1r (Miller et al.,1997). Attractin is a large, widely expressed transmembrane protein that acts as a low-affinity receptor for ASP (He et al.,2001) and is absolutely required for Agouti signaling. Mahogunin is also required for Agouti signaling but much less is known about its actions. This protein is an intracellular RING domain protein (a zinc finger with two sites that coordinate zinc), which exhibits E3 ubiquitin ligase activity in vitro (He et al.,2003). The model proposed to explain the action of these proteins is that, in the hair follicle, ASP signals through Attractin, leading to the Mahogunin-mediated ubiquitination of unknown target genes, resulting in their degradation (He et al.,2003; Fig. 4). One possible target would be the Mc1r receptor itself, which, when degraded would result in reduced eumelanin production at the time of agouti signaling. This explanation would be consistent with the epistatic relationships between the agouti, Mc1r, mahogany, and mahoganoid mutations. Alternatively, the ubiquitination may affect unknown components of the pathway leading to pheomelanin synthesis. Clearly, several steps in this signaling pathway are missing. Because several coat color mutations that affect this process remain to be cloned, including the dorsal-dark stripe and umbrous mutations, we can expect further insights and information in the future.

Figure 4.

Regulation of pigment type switching. The type of pigment synthesized by the melanocyte is regulated by several genes, most notably the α-MSH, Mc1r, Agouti, Mahogunin, and Attractin proteins. The α-MSH protein binds to the Mc1r receptor, leading to the activation of the cAMP pathway and resulting in eumelanin synthesis. The Agouti signal protein is temporally expressed in the hair follicle, interacts with the Mc1r and Attractin proteins, and inhibits the cAMP pathway, leading to pheomelanin synthesis. The Mahogunin protein plays a role in this pathway and may further enhance the inhibition of the cAMP pathway through ubiquitin-mediated degradation, for example, of the Mc1r protein (labeled as Ubi?).

Dark Skin Mutations

The mutations we have discussed so far affect hair color. In mice, melanocytes are mostly confined to the hair follicles in the epidermis, with the exception of ears, footpads, and the tail. In humans, however, melanocytes are dispersed among epidermal keratinocytes in addition to residing in the hair follicle. Until recently, few mouse models were available for studying the genetics of skin color. Large-scale mutagenesis screens, however, have now identified a large class of dark skin pigment mutations that has resulted in novel insights into skin color in mice (Fitch et al.,2003; Van Raamsdonk et al.,2004). These mutations increase either dermal or epidermal pigmentation (Fitch et al.,2003). Three of the four in the dermal class of Dark skin mutations (two different complementation groups, namely Dsk1 and Dsk7) affect the Gnaq and Gna11 genes, which encode two related Gα subunits, proteins that mediate the signal from G-protein coupled receptors (Van Raamsdonk et al.,2004). The mutations are gain-of-function mutations that result in overstimulation of G-protein signaling. In the mutants, there is an excess of melanoblasts during early development. Although a normal number of melanoblasts end up in the epidermis and hair follicles, the excess melanoblasts remain in the dermis, possibly due to their inability to traverse the basement membrane or due to the regulation of this process. How this is regulated is not known at present, but the existence of additional Dark skin mutations will hopefully allow the further molecular dissection of this interesting process.

PERSPECTIVES

Are Coat Color Mutations Ready for Systematic Approaches?

From the discussion above, it should be clear that the mouse coat color mutations have illuminated various different processes in melanocyte development and function. The cloning and molecular characterization of the additional known mutations will undoubtedly add further knowledge to our understanding of this important model cell type. In addition, novel coat color mutations are likely to arise from the numerous mouse mutagenesis screens currently in progress as well as from the many knockout mutations that will be generated in the future. The mutagenesis screens that have been performed so far are unlikely to have saturated the genome for coat color effects. Also, it is our experience that randomly mutagenizing the genome is more likely than knockout mutations to reveal subtle and unexpected effects of genes. Thus, additional mutagenesis screens, aiming for saturation of coat color genes, need to be performed to further characterize the biology of melanocytes. The example of dsu suggests that screening for suppressor mutations may also add important information. In addition to this strategy, recently developed technologies for modifying the mouse genome using bacterial artificial chromosome (BAC) recombineering has opened new avenues of investigation (Warming et al.,2005). For example, it is now possible to use BAC recombineering to mutate particular coat color genes in a BAC clone containing the gene and then test the effects of the mutations by generating transgenic mice. Thus, it is possible to dissect in detail the structure–function relationship of particular genes in the mouse, and because coat color is a sensitive indicator of function, this will provide a sensitive in vivo assay.

The recently available information on the mouse, human, rat, and other animal genomes, together with various different genome-wide methods that have been developed recently, will allow a new level of analysis of the melanocyte. Data from melanocytes, generated using the various genome-wide methods, will in the future be complemented with the available phenotypic information of coat color mutations, making the melanocyte an even more important model for function and development than it currently is. Melanocyte and melanoblast cell cultures are available from wild-type and many coat color mutant cells as well as from melanoma cells. Using gene expression and proteomic analysis, it is possible, therefore, to profile gene and protein expression in these different cell lines and developmental stages, with respect to gene and protein expression. Such analysis of gene and protein expression in wild-type melanoblasts and melanocytes will reveal which genes are active at each stage of development. Similarly, the analysis of gene expression in the different mutant melanocyte cell lines, as well as in melanoma cells, before and after differentiation, will give information about pathways and interactive networks involved in important decision-making processes.

Multiple transcription factors are known to affect pigment cells, including Mitf, Pax3, Sox10, and Snai2. In addition, it has been shown that β-catenin is important for normal melanocyte development (Hari et al.,2002). Overlaps and interactions in transcriptional regulation by these factors have been reported, as explained above. By using chromatin immunoprecipitation on a chip (ChIP chip) studies, it is now possible to dissect the target genes of each transcription factor at different stages of melanocyte development to determine the number and nature of target genes as well as to determine whether there are overlaps in the target genes of the different factors. Are there perhaps networks of transcription factors involved in transcribing melanocyte-specific genes at different stages of development? Do the transcription factor binding sites overlap in all or some cases? Is there a melanocyte-specific transcriptome? How does transcription factor function change with time and how does cell signaling affect the selection of binding sites? This kind of analysis will result in detailed information on the different genes regulated by these transcription factors at different times of development. In addition, this will reveal transcriptional networks where two or more transcription factors are involved in regulating sets of genes at certain times of melanoblast and melanocyte development.

It is our belief that, in the future, genome-wide methods such as these will be used to systematically dissect the development and function of the melanocyte. This will complement and enhance current knowledge about this cell type and, together with novel coat color mutations generated in future mutagenesis screens, result in a better understanding of the melanocyte than is possible for any other cell type in the organism.

Acknowledgements

We thank Dorothy C. Bennett for insightful comments on the manuscript and Allen Kane for the illustrations. E.S. was funded by the Science Fund of Iceland, the Science Fund of the University of Iceland. N.G.C. and N.A.J. were funded by the Intramural Research Support Program of the NIH, National Cancer Institute, Center for Cancer Research.

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