Because mouse strains with coat color variations harbor mutations that influence melanosomes, these mutations may also affect melanosomes of the iris and give rise to abnormal ocular phenotypes. To test this, we utilized an ophthalmic slit-lamp to examine cohorts of mice for ocular phenotypes. Our screen utilized two types of slit-lamp illumination, broad beam and transilluminating illumination. With broad beam illumination, a wide beam of light is shone at an angle across the surface of the eye, highlighting surface morphology. With transilluminating illumination, a small beam of light is shone directly through the pupil and the eye is assessed for light reflected off inner surfaces of the back of the eye. In healthy irides, reflected light is blocked by the iris and the iris appears black. In thin, depigmented, or diseased irides, reflected light passes through areas of the iris and appears as areas of red (some light passing through) or white light (greater amounts of light passing through). Using these techniques, we performed an analysis comparing the iris of wild-type mice to several mutant strains.
Iris phenotypes of wild-type C57BL/6J mice
The iris of C57BL/6J mice is densely pigmented with a complex morphology (Figure 1). The iris appears uniformly deep sienna-brown in color with a smooth surface accentuated by a number of small vessels lying near the surface. The pupil is round, bordered by a beaded concentric ring of pupillary rough, and often contains a small notch inferiorly (Smith et al., 2000). As expected for densely pigmented intact irides, the iris appears solid black when viewed by transilluminating illumination. These features showed little visible variability between different individuals or sexes.
Figure 1. Normal iris phenotypes of wild-type C57BL/6J mice. Comparisons of a representative 3-month eye (left column) to an 18-month eye (right column). (A) The normal C57BL/6J iris of young mice as viewed with broad-beam illumination originally imaged at 25× magnification. The iris is characterized by a sienna-brown color, a complex surface morphology with several small underlying vessels, and a circular pupil. The bright white circle to the left of the pupil is a reflection from flash photography. (B) With age, a number of clump cells are present on the surface of the iris and the iris becomes slightly more reddish in color. (C, D) At higher 40× magnification and less image reduction, clump cells are more readily visible, each casting a characteristic small crescent shadow (several are indicated by open white arrows, others in the field are unmarked). (E, F) Unstained cryosections of the same eyes shown above showing the presence of a melanin engulfed phagocytic clump cell in panel F on the surface of the iris stroma as a normal consequence of aging. These cells were not visible in 0/64 sections from the young eye in E and were present on the iris in 6/70 sections analyzed from the aged eye in F. Scale bar = 50 μm.
Download figure to PowerPoint
As C57BL/6J mice aged, two changes occurred (Figure 1B, D, F). First, iris color changed, gradually transitioning from a slightly darker hue in young mice toward a more reddish hue with age. Second, small amounts of dispersed pigment became visible. These typically appeared as rounded cells on the iris surface and likely correspond to the phagocytic clump cells described by Koganei (Wobmann and Fine, 1972). In mice 2–6 months in age, dispersed pigment was rare (one eye exhibited pigment on the cornea, 59 others were normal). In mice older than 6 months, the presence of a small number of clump cells on the iris surface was nearly universal and tended to become more prominent with increasing age (n = 12 mice 6–9 months, 15 mice 9–12 months, and 15 mice 12–15 months). Transillumination defects remained absent with age. Combined, these results indicate that small amounts of pigment dispersion are a normal feature of aging on the C57BL/6J genetic background, but C57BL/6J mice otherwise maintain an intact healthy iris into advanced age.
Iris phenotypes among a survey of 12 mouse substrains with coat color variations
We next analyzed a diverse group of 11 substrains with coat color variations or mutations that otherwise influence pigmentation (Figures 2–4, summarized in Table 1), all with an identical genetic background (C57BL/6J). For simplicity, we will refer to each of these by their original allele names (see Materials and methods for full nomenclature). From observations of these strains, an interesting dichotomy became apparent; some alleles have apparently discordant effects on coat color and iris phenotypes, whereas others are quite similar.
Figure 2. Substrains exhibiting a correlation between coat and iris appearance. Coat color (left column), broadbeam illumination of iris (middle column), and transilluminating view of iris (right column). The bright white circle (to the left of the pupil with broad-beam illumination and central with transilluminating illumination) is a reflection from flash photography. (A–C) Wild-type C57BL/6J mice have darkly pigmented coats and darkly pigmented irides that are sienna-brown in color. Because the irides are darkly pigmented, the iris appears black with transilluminating illumination. (D, E) albino mice have coats and irides totally lacking melanin pigment. (G–I) pallid mice have severely diluted coats and irides. All mice were homozygous for the indicated mutations and were maintained on a C57BL/6J genetic background.
Download figure to PowerPoint
Figure 3. Substrains exhibiting discordant coat color and iris appearances. Coat color (left column), broadbeam illumination of iris (middle column), and transilluminating view of iris (right column). (A–C) beige mice have slightly diluted coats, but irides that appear very dark. (D–F) buff mice have relatively severe dilution of coat color, but only a slight dilution of iris pigmentation evident by mild transillumination defects. The transillumination defects exhibited no discernable pattern, other than that the extent of the defects tended to increase peripherally. No influence of age was detectable. (G–I) cocoa mice have a modest dilution of coat color but strikingly hypopigmented irides. (J–L) recessive yellow mice have yellow coats, but normally pigmented irides that are indistinguishable from those of normally pigmented C57BL/6J mice. All mice were homozygous for the indicated mutations and were maintained on a C57BL/6J genetic background.
Download figure to PowerPoint
Figure 4. Substrains with mild, or no, change in iris appearance. Coat color (left column), broadbeam illumination of iris (middle column), and transilluminating view of iris (right column). (A–C) chocolate mice have a modest alteration in coat color and mild transillumination defects that tended to increase peripherally. These transillumination defects did not worsen with increasing age among the mice examined. In contrast, other strains with similarly modest alterations in coat color maintain completely normal irides; including, (D–F) leaden mice have slightly diluted coats and normal appearing irides (the leaden substrain utilized here also carried the fuzzy allele, so it can additionally be concluded that fuzzy does not influence the iris), and (G–I) gunmetal mice have slightly diluted coats and normal appearing irides. Two substrains with mutations that do not influence adult coat color at all, but do influence melanosomes in other tissues were also examined; including, (J–L) as adults, pale ear mice have normal coat color but irides that appear slightly darker (note that the ears and tail of pale ear mice are also light colored), and (M–O) shaker 1 mice have normally colored coats and normal appearing irides. All mice were homozygous for the indicated mutations and were maintained on a C57BL/6J genetic background.
Download figure to PowerPoint
Table 1. Summary of iris phenotypes survey
|Strain name||Symbol||Gene||Background||Total no. mice||Oldest (months)||Comments|
|beige||Lystbg-J||Lysosomal trafficking regulator||C57BL/6J||13||10||Very dark iris and pigment dispersion|
|buff||Vps33abf||Vacuolar protein sorting 33A (yeast)||C57BL/6J||7||5||Mild transillumination increasing peripherally|
|chocolate||Rab38cht||Rab38, member of RAS oncogene family||C57BL/6J||11||12||Mild transillumination increasing peripherally|
|cocoa||Hps3coa||Hermansky-Pudlak syndrome 3 homolog||C57BL/6J||12||7||Uniformly hypopigmented|
|gunmetal||Rabggtagm||Rab geranylgeranyl transferase, a subunit||C57BL/6J||7||5||Wild-type|
|light||Tyrp1B-It||Tyrosinase related protein 1||LT/SvEiJ||46||22||Pigment dispersion|
|nm2798||DctSIt-It3J||DOPAchrome tautomerase||C57BL16J||18||19||Pigment dispersion|
|pale ear||Hps1ep||Hermansky-Pudlak syndrome 1 homolog||C57BL/6J||9||6||Slightly dark iris|
|pallid||Pldnpa||Pallidin||C57BL/6J||9||6.5||Near total albinism|
|recessive yellow||Mc1re-J||Melanocortin 1 receptor||C57BL/6J||7||5.5||Wild-type|
|shaker 1||Myo7ash1−8j||Myosin VIIa||C57BL/6J||9||7||Wild-type|
|silver||Sisi||Silver||a Tyrp1bSisi/J||15||24||Wild-type, does not alter Tyrp1b|
|vitiligo||MitfmI-vit||Microphthalmia-associated transcription factor||C57BL/6J||12||21.5||Pigment dispersion|
Some substrains exhibited a correlation between coat and iris appearance. For example, wild-type C57BL/6J mice have dark coats and dark irides (Figure 2A–C), albino mice completely lacking coat pigmentation also completely lacked iris pigment (Figure 2D–F), and pallid mice with severely diluted coat color exhibited severely diluted irides (Figure 2G–I). These results indicate that some alleles indeed have similar effects on pigmentation of the mouse iris and coat, likely because they represent key molecules that are absolutely required for melanin synthesis of any type.
However, coat color did not always predict iris appearance. This was strikingly observable in three different comparisons: (1) the slight dilution of coat color in beige mice is similar to many other substrains, but the iris appears very dark (Figure 3A–C). Eyes of beige mice also exhibited a unique pattern of transillumination defects and pronounced pigment dispersion. (2) The buff substrain exhibits a relatively severe dilution of coat color, but only a slight dilution of iris pigmentation (Figure 3D–F). Conversely, the cocoa substrain exhibits only a modest dilution of coat color, but pronounced iris transillumination defects (Figure 3G–I). (3) The recessive yellow substrain exhibits yellow coats, but irides of a color and morphology indistinguishable from wild-type C57BL/6J mice with black coats (Figure 3J–O). This result also offers further in vivo support for the prior finding that mouse iris melanocytes are insensitive to signaling pathways modulated by α-melanocyte-stimulating hormone (Li et al., 2006). Combined, these results identify alleles with distinguishable influence to the coat and iris, perhaps because they disrupt processes of differing importance to melanosomes in the unique context of each tissue.
Some substrains had irides with subtle, or no, changes in appearance. One substrain with modest alterations in coat color (chocolate) exhibited relatively mild transillumination defects (Figure 4A–C); two others with similarly modest alterations in coat color (leaden and gunmetal) maintained completely normal irides (Figure 4D–I). These observations correlate well with a recent report of ocular phenotypes observed in chocolate mice (Brooks et al., 2007). These results indicate that not every mutation influencing coat color will necessarily visibly influence the iris.
Two substrains with mutations that do not influence adult coat color at all (pale ear and shaker 1), but do influence melanosomes in other distinct populations of pigmented cells were also examined (Futter et al., 2004; Nguyen and Wei, 2007). Irides of pale ear mice exhibited a subtle phenotype characterized by a slight darkening across the surface of the iris stroma (Figure 4J–L). In contrast, eyes of shaker 1 mice maintained completely normal irides (Figure 4M–O). These results indicate that mutations influencing melanosomes, including mutations that do not influence coat color, can also give rise to abnormal iris phenotypes.
From these examinations, an interesting observation regarding iris color became apparent. It has long been appreciated that mutations influencing melanin color of the coat also influences melanin color of the iris (Pierro, 1963). We were, therefore, surprised to find that the majority of substrains exhibited similar sienna-brown iris colors. Given the wide range of coat colors represented within the screen, this again emphasizes that there are differences in the pathways regulating pigmentation of the coat versus iris.
Iris phenotypes among mutant mouse strains genetically associated with Tyrp1
We have been particularly interested in identifying mouse strains exhibiting pigment dispersion (Anderson et al., 2001, 2002, 2006). Here, we use ‘pigment dispersion’ to indicate the phenotype characterized by any aberrant deposition of pigment throughout the anterior chamber of the eye. The pigment may consist of dispersed melanin pigment itself or pigment that has become engulfed by other cells. In humans, pigment dispersion is a primary feature of pigment dispersion syndrome and can also occur in pseudoexfoliation syndrome, intraocular melanoma, and uveitis (Ball, 2004; Ritch et al., 1996; Shuba et al., 2007; Sowka, 2004). Because all of these diseases are of substantial medical importance, the identification of mouse strains exhibiting pigment dispersion could be of substantial benefit.
In addition to the identification of pigment dispersion in beige mice reported above, forms of pigment dispersion in mice have thus far only been observed in a small number of strains (Anderson et al., 2001, 2002, 2006; Boissy et al., 1987; Collier et al., 1984; John et al., 1998; Marneros and Olsen, 2003; Rodemer et al., 2003). Among these, the form of pigment dispersion occurring in DBA/2J mice is of particular relevance to pigment-related genes. DBA/2J mice develop a pigment dispersing iris disease as a consequence of mutations in two genes that both encode melanosomal proteins, Tyrp1b (brown) and GpnmbR150X (Anderson et al., 2002; John et al., 1998). Iris phenotypes of congenic strains containing either the Tyrp1b or GpnmbR150X mutations within the C57BL/6J genetic background have also recently been described in detail (Anderson et al., 2006). It is unknown whether pigment dispersion might also occur in strains with different alleles of these genes or in strains with other mutations in associated genetic pathways. While relatively little is currently known concerning the functions of Gpnmb in pigment-producing cells of the mouse, Tyrp1 has been more extensively studied and multiple mouse strains relevant to Tyrp1 exist. Some of these, such as albino (genetically upstream of Tyrp1) and chocolate (targeting of TYRP1 to melanosomes), were already examined in the above experiments (Figures 2 and 4, respectively). In addition to those, we examined more strains genetically associated with Tyrp1; LT/SvEiJ mice carrying the Tyrp1B-lt mutation, vitiligo mice carrying the Mitfmi-vit mutation, and silver mice.
Inbred LT/SvEiJ mice are homozygous for the Tyrp1 allele, light, which acts dominantly to influence melanocyte survival and coat color (Johnson and Jackson, 1992; Mac, 1950). To test whether the previously described iris disease associated with Tyrp1 is specific to only the brown allele (Anderson et al., 2002; Chang et al., 1999; Libby et al., 2005), we analyzed the eyes of LT/SvEiJ mice (Figure 5A–C). Pigment dispersion was apparent in some eyes of young LT/SvEiJ mice (few specks of dispersed pigment within the pupil in 4/36 irides of 1–6 months mice) and became increasingly common and severe with age (specks of dispersed pigment present in pupil of 8/18 irides of 6–12 months mice, 13/24 irides of 12–18 months mice). In advanced age, abundant dispersed pigment was present in almost all eyes (specks and accumulations of dispersed pigment present in pupil or on cornea in 10/12 irides of 21-month mice; Figure 5B). In mice over 12 months of age, mild small foci of iris transillumination defects became visible (data not shown). Several other non-age-related ocular abnormalities occurred in light mice, including mild lens opacities (all eyes), lens-corneal adhesions (12/92 eyes), misshapen pupils (34/92 eyes), and dysmorphic pupillary rough (8/92 eyes). With respect to our current experiments, the most relevant of these findings pertains to pigment dispersion; iris disease associated with Tyrp1 mutation is not a specific consequence of the brown allele.
Figure 5. Pigment dispersion is a common, but not universal, feature of mouse strains with mutations related to Tyrp1. Comparisons of coat color (left column) and ocular phenotypes (right columns). (A) The light allele in LT/SvEiJ mice results in the production of hair that is pigmented at the tip, but very lightly or not pigmented along the hair shaft. As a consequence, coat color is rapidly lightened as hair lengthens. (B) Pigment dispersion was commonly present in the eyes of aged light mice. (C) Higher magnification image of same pupil shown in panel B. Several clumps of dispersed pigment are clearly visible in the pupil (one prominent example indicated with arrowhead, several other unmarked also visible). (D) The vitiligo allele results in a coat that is initially lighter than normal, with extensive white spotting (front mouse). With increasing age, the coat becomes progressively whiter due to increasing numbers of white hairs with each molt (back mouse). (E) Pigment dispersion was striking in eyes of aged vitiligo mice, as shown here in the pupil (arrowhead), across the surface of the iris (open white arrow), and in a pronounced pool accumulated inferiorly (dark band marked by solid black arrow). (F) Eyes of several vitiligo mice became severely enlarged, as sometimes occurs in mice with glaucoma. (G) The silver allele results in a mix of normal and hypopigmented hairs, which together cause a characteristic silvering of the coat. Heterozygosity for Tyrp1b enhances the silver coat color phenotype as shown here among three young silver homozygotes with differing Tyrp1 genotypes (Tyrp1+/+ front, Tyrp1b/b left, and Tyrp1b/+ right). (H) Mice heterozygous for Tyrp1b and homozygous for Sisi maintain healthy irides lacking pigment dispersion. (I) Mice homozygous for both Tyrp1b and Sisi exhibit a characteristic Tyrp1 mutant iris phenotype characterized by iris atrophy and pigment dispersion. Neither the onset nor the severity of these Tyrp1b-mediated phenotypes were influenced by Si. Note, because of the unique photographic flash settings used to capture the pigment accumulation near the highly reflective sclera in panel E, the apparent color of this iris is not directly comparable to the images in other panels.
Download figure to PowerPoint
The Mitf gene encodes a transcription factor previously demonstrated to regulate expression of several genes important to ocular development and function, including Tyrp1 (Fang et al., 2002; Murisier and Beermann, 2006; Smith et al., 1998). Whereas, some mutant alleles of Mitf in mice result in the presence of micro-ophthalmic or albino eyes (Steingrimsson et al., 2004), the vitiligo allele allows grossly normal ocular development and pigmentation to initially occur and is an interesting candidate for exhibiting pigment dispersion. To test this possibility, we performed slit-lamp examinations on the vitiligo strain of mice (Figure 5D–F). Dispersed pigment became evident by 7–8 months (single specks of dispersed pigment present in pupil of 2/18 eyes). Past 12 months of age, dispersed pigment was readily apparent across the iris surface, in the pupil, and on the cornea of all mice (Figure 5E). The distinctly rounded appearance of the dispersed pigment suggests that it likely correlates to pigment engulfed macrophages previously observed in histologic sections of vitiligo eyes (Boissy et al., 1987). Interestingly, several eyes and anterior chambers with pigment dispersion also became enlarged (6/30 eyes of 15–21 months mice), suggesting that the accumulation of pigment within these eyes may be raising intraocular pressure (Figure 5F). No transillumination defects were observed. The occurrence of pigment dispersion in these mice indicates that at least some genes in genetic pathways influencing Tyrp1 can also contribute to the pigment dispersion phenotype.
Interestingly, the iris phenotype observed in vitiligo mice was more severe than previously observed Tyrp1 mutant phenotypes (Figure 5; Anderson et al., 2006, 2002). This observation suggests that in addition to regulating Tyrp1 expression, MITF may transcriptionally regulate additional target genes impacting iris integrity. A key candidate for such a factor is Gpnmb. In mice, mutation of Gpnmb causes significant pigment dispersion (Anderson et al., 2002, 2006) and experiments in quail have functionally associated an ortholog of Gpnmb, QNR-71, as a likely target of MITF (Aksan and Goding, 1998; Turque et al., 1996). As a step toward determining whether MITF regulates Gpnmb in mice, we examined the immediate flanking region of the Gpnmb transcription start site for potential MITF binding M-box sequences (Aksan and Goding, 1998). A consensus M-box sequence (5′-TCATGTG-3′) was identified at −34 bp relative to the Gpnmb transcription start site (M-box starting at NCBIM37:6:48986590; based on the transcription start site indicated by Gpnmb mRNA RefSeq NM_053110). A similar M-box-related sequence is also located at −96 bp of the mouse gene and both elements appear present at similar positions 5′ of the human GPNMB gene. This observation supports the hypothesis that Gpnmb may be a downstream target of MITF. Further experiments will be required to test this directly.
The Si gene encodes a close homolog of Gpnmb (Shikano et al., 2001; Weterman et al., 1995). Interestingly, both Gpnmb and Si interact genetically with the Tyrp1b mutation (Anderson et al., 2002; Theos et al., 2005). Our initial studies involved silver mice with a mixed genetic background in which the Tyrp1b allele was also segregating (Figure 5G–I). In mice homozygous for Sisi and heterozygous for Tyrp1b, pigment dispersion was not detected through advanced age (14 mice aged 10–22 months examined; Figure 5H). In mice homozygous for both Sisi and Tyrp1b (17 mice aged 10–31 months), eyes developed iris atrophy typical for mice carrying the Tyrp1b mutation (Anderson et al., 2001, 2006; Chang et al., 1999). The silver allele had no influence on the age of onset nor severity of these phenotypes and no other ocular phenotypes were present (Figure 5I). Similar results were obtained with mice from the silver stock cryopreserved by The Jackson Laboratory (STOCK a Tyrp1bSisi/J, data not shown). These results indicate that despite the evidence linking Si to the pigment dispersion-related Tyrp1 and Gpnmb genes, the silver allele does not influence pigment dispersion.
Combined, these results indicate that mutations influencing Tyrp1 are a common, but not universal, cause of iris disease. As explained below, a central relevance of this genetic pathway to pigment dispersion was further supported by our analysis of a new spontaneous coat color mutant strain exhibiting pigment dispersion.
Iris phenotypes in the nm2798 strain of mice
The nm2798 mutation is a spontaneously occurring coat color variant isolated at The Jackson Laboratory causing a semi-dominant effect on coat color whereby a normally black coat appears light grey in homozygotes and dark grey in heterozygotes (Figure 6A). Because of our interest in relationships between coat color and iris phenotypes, we screened this strain for potential iris phenotypes (n = 5 mice at 1 month, 12 mice at 4–6 months, 2 mice at 8 months). Irides of nm2798 homozygotes had a healthy appearance through 5 months (Figure 6B). After 8 months, all nm2798 homozygotes showed signs of iris pigment dispersion. In follow-up analysis of aged mice (six homozygous mice aged 14–19 months), all eyes exhibited pigment dispersion across the iris surface and mild transillumination defects (Figure 6C,D). Similarly aged cohorts of nm2798 heterozygotes maintained healthy irides lacking pigment dispersion and transillumination defects.
Figure 6. Phenotypes and sequence analysis of the spontaneously occurring nm2798 mutation. (A) The nm2798 mutation acts semi-dominantly to influence coat color, as shown here in a comparison of a heterozygote (front) and homozygote (back). (B) Healthy iris of a young nm2798 homozygote. (C) Aged nm2798 homozygote showing dispersed pigment across the surface of the iris and (D) mild transillumination defects spread across the iris. (E) Higher magnification view with a narrower slit of illumination showing the same iris as in panels (C, D). The discretely rounded appearance of the dispersed pigment along the iris surface (three examples marked with open white arrows, several others in field unmarked) indicates that it is likely present within phagocytic clump cells. Pigment is also accumulating inferiorly (solid black arrow). (F) Sequence comparison of wild-type (B6) versus mutant (nm2798) alleles. A single base pair substitution within exon 8 of the Dct gene results in a mis-sense mutation changing a glycine to arginine (asterisk). The base pair and amino acid changes are both identical to previously described slaty light mutation. The change occurs within the predicted transmembrane spanning domain (TM). Other motifs of the DCT protein include a signal sequence (SS); cysteine-rich domains (CYS-rich); and metal-binding motifs (MeA, MeB).
Download figure to PowerPoint
Genetic crosses between nm2798 and CAST/EiJ mice were utilized to map the nm2798 mutation to chromosome 14, closely linked to D14Mit42 (data not shown). Because the Dct gene is located near this position and the nm2798 coat color resembles slaty variants, a candidate gene approach was utilized to analyze Dct for possible mutations. A single G to A base change resulting in an amino acid substitution within the predicted transmembrane encoding portion of the DCT protein was identified (Figure 6F). All mice exhibiting the homozygous coat color phenotype were homozygous for the change, and all mice exhibiting the heterozygous coat color phenotype were heterozygous for the change (n = 6 mice of each genotype). No other changes were detected, indicating that the G to A change is highly likely to be the disease causing mutation.
Dct (also referred to as Tyrp2) encodes a transmembrane melanosomal protein sharing significant homology with TYRP1 (Budd and Jackson, 1995). DCT influences melanin synthesis and melanosome morphology (Hirobe and Abe, 2007; Matsunaga and Riley, 2002; Solano et al., 2000a; Winder et al., 1994). Interestingly, the exact mutation identified in nm2798 has been previously identified as the cause of the slaty light phenotype in a completely distinct strain of mice (precisely the same nucleotide change resulting in precisely the same amino acid substitution) (Budd and Jackson, 1995). Accordingly, we propose to hereafter refer to this allele as slaty light 3J. This mutation represents a slightly unusual case whereby the exact same genetic mutation has spontaneously arisen multiple times. Apparently, this amino acid position plays a critical role in enzyme function, one that is perhaps uniquely disrupted by this substitution.