Chapter 6

Beige, Silver, Greying with Age, and Other Determinants

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I. Beige (bg)

For the bg allele:
bg Allele (MGI) Gene (MGI) All Alleles (MGI)

Beige [bg; chromosome 13 ( Lane, 1962)] is a recessive mutation affecting both coat and eye color. The mutation is of particular interest because it produces a syndrome strikingly similar to Chediak-Higashi syndrome in man ( Blume at al., 1968; Blume and S. Wolff, 1972). 1 This syndrome is characterized by an autosomal recessive inheritance, pigment dilution, large melanin granules, large lysosomal granules, and an increased susceptibility to infections. 2 Its effects on pigmentation have been studied by Windhorst and her associates (Windhorst et al., 19661968; Zelickson et al., 1967). 3

A. Origin

Beige was reported first by Kelley in 1957 after it was probably radiation induced at Oak Ridge. However, it also occurred at Brown University in 1955 as a spontaneous mutation in Chase's YZ57/Ch strain ( Chase, 1959), a mutation that was known as "slate" ( slt) until its allelism with bg was demonstrated ( Lyon and Meredith, 1965b; see Chase, 1965). This mutation is currently referred to as bgslt. Two mutations to beige have occurred also at the Jackson Laboratory, one in a yellow stock and the other in strain C57BL/6J ( Lane, 1962) ( Plate 3-A). The mutation is maintained on two backgrounds; an inbred derivative of the heterogeneous stock in which it occurred at Oak Ridge, known as SB/Le ( Lane and E. Murphy, 1972) and C57BL/6J ( bgJ).

B. Influence on Pigmentation

In the heterogeneous agouti stock in which it was first reported, the eye color of bg/bg mice was light at birth and varied from ruby to almost black in adults. A/—;B/—;bg/bg mice display reduced ear and tail pigmentation, and the coat is lighter than the coat of wild type mice, particularly at the base of the hairs ( Kelly, 1957). This basal dilution, which is first noticeable at about 15 days of age, is somewhat variable, and tends to become darker in older animals ( Kelly, 1957). The overall appearance of A/—;b/b;bg/bg animals is "cafe-au-lait" and their eyes at birth are lighter than either A/—;b/b;Bg/— or A/—;B/—;bg/bg genotypes. Moreover, the relatively greater dilution effect on the base of the hair is not as pronounced in brown beige as in black beige mice ( Kelly, 1957).

A more detailed account of the effect of beige on pigmentation is given by Pierro and Chase ( 1963) in their description of slate. This mutation was maintained in a stock segregating for yellow ( Ay/a) and piebald spotting (s) and it was found that ruby-red eyes were restricted to bgslt/bgslt;s/s mice; bgslt/bgslt;S/— and Bg/—;s/s animals had dark eyes. These observations, therefore, indicate some interaction between beige and piebald in the variability in eye color.

a/a;B/B;bgslt/bgslt mice have a slate black coat with the hairs displaying a considerable amount of medullary and cortical pigment ( Figure 6-1a). The pigment in the medulla of the hair was generally very highly clumped, but individual granules can be resolved in some septae. The dimensions of individual granules along any given axis varied from 0.5 to almost 4 microns. 4 Some granules were long ovals but irregular shapes were common, especially among the larger pigment masses (compare Figure 6-1c with d). Some variation was also observed among cortical granules which were unusually long ovals measuring about 1 micron long and about half as wide. However, some granules were compressed laterally and elongated to as much as 2-3 microns, while others were almost as wide as they were long ( Pierro and Chase, 1963).

C. Interaction with Other Determinants

Pierro and Spiggle ( 1970) found that the characteristic exclusion of eumelanin granules from the proximal region of the hair shaft of Blt (light) animals (see Chapter 3, Section I, G) did not occur in Blt mice homozygous for bgslt. 5 On the other hand, phaeomelanin was eliminated from the proximal portion of the hair shaft in yellow, beige ( Ay/a;bgslt/bgslt) animals. Initially this situation was found to occur only during the later hair growth cycles in a small number of animals, but when selected for it resulted first with a lightening of the coat after the completion of the second hair growth cycle, and subsequently with the absence of yellow granules in the proximal portion of hairs of the initial pelage. 6 Nevertheless, melanocytes appeared to be present in hair follicles throughout the cycle.

According to Pierro and Chase ( 1963) when beige is combined with dilute ( a/a;bgslt/bgslt;d/d) a "silvery-blue" coat color results and the eyes are black, or reddish if the animals are piebald. Under the light microscope cleared beige dilute hairs displayed an almost complete absence of pigment from approximately the distal third of the hair shaft, but the more proximal regions were as heavily pigmented as the hairs of nondilute beige mice.

Beige ruby-eye ( a/a;bgslt/bgslt;ru/ru) mice have an off-white coat and pink eye color. The cleared hairs from these mice "showed that cortical pigment was absent, and that many medullary septae had no pigment granules at all." Melanin granules were found in more medullary septae in the proximal regions of the hair shaft than in the distal portion. The granules themselves displayed the same characteristics as those in beige hairs, but were very few in numbers ( Figure 6-1b).

The coats of animals homozygous for bgslt and pink-eyed dilution ( p) are only slightly lighter than those of mice homozygous for p alone. The eyes of these mice are of course pink. Cleared hairs revealed that cortical pigment was absent. A few medullary septae lacked pigment granules, but these were restricted to the more distal regions of the hair shaft. In general, the pigment granules themselves were very much like those found in p/p mice, but in a few instances larger than expected granules were observed ( Pierro and Chase, 1963).

It thus appears that in animals homozygous for both beige and ruby-eye the medullary granules of the hair display the characteristic features of beige granules, whereas in pink-eyed beige mice the medullary granules generally display the features characteristic of p granules. These observations indicate that at least insofar as the morphology of the pigment granules of the hair is concerned, and, as noted below, those of the retina as well ( Hearing et al., 1973), beige is epistatic to ru but hypostatic to p ( Pierro and Chase, 1963). Nevertheless, some interaction between beige and both ruby-eye and pink-eyed is indicated by the absence of cortical pigment in both double heterozygotes, as well as by the extreme reduction in medullary pigment in bgslt/bgslt;ru/ru mice ( Pierro and Chase, 1963)

Although beige, like dilute ( d/d) and leaden ( ln/ln), appears to bring about the clumping of medullary pigment granules, it has no effect on melanocyte morphology. Presumably, therefore, this clumping effect must have a different basis.

D. Development of Pigment Granules

In a more detailed analysis of the effect of beige on melanin synthesis Pierro ( 1963b) studied pigment granule genesis in epithelial, uveal, harderian gland, and hair bulb melanocytes of both normal and beige ( a/a;B/B and a/a;B/B;S/s;Bg/bg) mice. In the epithelial melanocytes of the retina (which becomes pigmented at about the eleventh day of gestation) of normal animals he found the first pigment granules to be relatively small and uniform spheroids which approximately doubled in size during the first 24 hours after their initial appearance. As the number of these granules increased their sizes and shapes became more variable: first long ovals, then rod and spindle shaped granules appeared ( Figure 6-2a). Although in beige mice the first granules observed in epithelial melanocytes were likewise spheroidal, and of a size similar to the granules in normal mice, fewer granules were found within each cell. Moreover, not only did the number of granules appear to remain fairly constant for some time, but their numbers subsequently seemed to decline. This occurred even though granule synthesis continued and the various granule types appeared. It soon became evident that this apparent decrease in the number of granules resulted from the fusion of individual granules to form "giant granules" ( Figure 6-2b). Thus in some cells "grape-like clusters and dumb-bell shaped granules" were found which appeared to represent intermediate stages leading to the formation of still larger masses. In fact, a number of cells were found in which only a single enormous pigment mass occurred ( Pierro, 1963b).

As far as the neural crest-derived melanocytes of the uveal tract and harderian gland were concerned, from their first appearance the pigment granules in these cells of normal mice were uniform long-ovals, considerably smaller than any of those found in epithelial melanocytes. Furthermore, unlike the granules of the retina, these did not undergo any apparent change. On the other hand, while the first granules to appear in beige uveal and harderian gland pigment cells were also long-ovals, there were not as many of them and, as progressively older mice were examined, their size increased significantly. Moreover, the larger they became the more irregular was their shape. Once again it seemed apparent that in at least some cases granule size increased at the expense of granule number, indicating that the large masses of pigment were due to the fusion of granules ( Pierro, 1963b).

The pigment granules of beige and nonbeige hair bulb melanocytes also were essentially similar in size and shape during the early stages (Anagen III and IV) of hair growth. In both genotypes, most of the granules were elongated ovals which did not exceed 0.5 microns in length. During later stages of hair growth (Anagen V and VI), however, there were marked differences. Not only were the pigment granules of normal hair bulbs much more numerous but they were much more uniform in size: small, long-ovals. Beige granules, on the other hand, showed considerable variation in size and shape even though a significant number of them were long-ovals approximately 1 micron in length ( Pierro, 1963b).

Taken together these observations indicate that there is a reduced number of pigment granules in the melanocytes of beige mice and that this reduction is due both to the synthesis of fewer granules and to the fusion of granules into progressively larger bodies. In the hair bulb this fusion of granules occurs in both the follicular melanocytes and the recipient matrix cells and since, as the granules become larger, their shapes become more irregular, all of these attributes — granule number, size and shape — contribute to the mutant's phenotype ( Pierro, 1963b).

E. Ultrastructural Studies

The ultrastructure of the melanosomes of nonagouti beige mice, as well as those from brown ( b/b), ruby-eye ( ru/ru), albino ( c/c), and pink-eyes dilute ( p/p), nonagouti beige donors, has been examined by Hearing and his associates ( 1973). As might be expected they found in both the choroid and retina of bgJ/bgJ mice that giant granules appear to be formed by the random aggregation of premelanosomes and melanosomes, and their subsequent asynchronous melanization. Thus unlike normal granule formation where melanin deposition proceeds more or less uniformly, several stages of melanization often were visible within a single beige granule. This asynchronous melanization of giant granules was observed also in the eyes of brown beige animals ( Figure 6-3a and b) where it was estimated that about 80% of all the granules in the adult choroid and retina were fused into giants. 7

In ruby-eye beige animals melanogenesis in the choroid was delayed until after parturition as it is when ruby-eye is present by itself. However, unlike the premelanosomes formed in the retina of these double mutants, where approximately 80% fused to form giant melanosomes, the granules of the choroid displayed no evidence of aggregating into giants and 95% remained particulate ( Hearing et al., 1973) (see Chapter 4, note 20). Thus for some reason, perhaps related to the delay in the onset of melanogenesis, in ru/ru;bgJ/bgJ mice, beige seems to affect the fibrillar retinal granules as well as the granules of the epidermal melanocytes (see above), but not the particulate choroidal ones.

In contrast to the effect of beige on b/b and ru/ru granules, it had no influence when combined either with c/c or p/p. In albino beige animals the premelanosomes in the retina and choroid were like those of nonbeige albino mice, and in bgJ/bgJ;p/p mice there was likewise no tendency for the melanosomes to aggregate into giant granules ( Hearing et al., 1973). 8

These ultrastructural observations suggest that the formation of giant granules in beige mice results from the fusion of individual, normally sized granules ( Figure 6-3c), primarily those in the premelanosome stage. Whether this fusion is caused by a membrane abnormality, a modification of melanofilaments, or some other abnormality is not known ( Hearing et al., 1973). However, the fact that the melanosomes of neither albino nor pink-eyed dilute animals are affected by beige raises the possibility that a threshold amount of tyrosine activity may be required before this mutant's effect in the melanocyte is expressed ( Hearing et al., 1973). In this regard it would be of interest to know how the other effects of beige (see Section F) are expressed in albino and pink-eyed dilute mice.

F. Other Effects

In addition to its effect on the pigmentation of the hair, beige has been reported to be sometimes associated with temporary hair loss ( Pierro and Chase, 1965). In the YZ57/Ch strain in which bgslt originated, about 5% of bgslt/bgslt mice lost most of their dorsal hairs of the first coat during the fourth week after birth, i.e., following the cessation of the first hair growth cycle. The second coat grown by these animals was normal although somewhat delayed in some cases. Coats of affected mice usually remained normal in appearance thereafter, but in some instances the coats of older animals became somewhat sparse ( Pierro and Chase, 1965). Breeding studies demonstrated that the penetrance and expression of this hair loss trait depended upon the genetic background and, since it could be readily selected for, that a small number of genes were involved. In fact the presence of p/p, d/d, and especially b/b, or genes closely associated with these coat-color determinants, seemed to increase greatly the number of beige animals which displayed the temporary hair loss condition.

Encouraged by Pierro and Chase's observation that beige mice possessed unusually large melanin granules, Lutzner and his colleagues ( 1967), who were searching for a small animal model for the Chediak-Higashi syndrome, examined the peripheral blood and bone marrow of these mice for giant leukocyte granules. Their search was not in vain as they found that lymphocytes, neutrophils, and eosinophils of all beige mice, but not of non-beige animals, possessed giant granules. Subsequently it was demonstrated, using acid phosphatase as a marker ( C. Oliver and Essner, 1973), as well as Sudan black and peroxidase stains ( J. Bennett et al., 1969), that these giant granules were enlarged (anomalous) lysosomes, often in the form of aggregates, and that they occurred not only in leukocytes ( Blume et al., 1969; C. Oliver and Essner, 1975) but in a variety of other tissues as well ( C. Oliver and Essner, 1973). The most anomalous lysosomes occurred in liver parenchymal cells, kidney proximal tubule cells, Purkinje cells, and granulocytes ( C. Oliver and Essner, 1973; Essner and C. Oliver, 1973, 1974). 9 Since there is evidence that in the Chediak-Higashi syndrome, too, there is a basic alteration in lysosomes ( S. Wolff et al., 1972), these findings further strengthened the homology between the beige mouse and this disease.

This homology was made even more apparent when it was established that on some genetic backgrounds, homozygous beige mice have a significantly higher incidence of pneumonitis than nonbeige animals ( Lane and E. Murphy, 1972). In the C57BL/6J strain, beige mice do not develop spontaneous pneumonitis whereas it does develop when the SB/Le background predominates ( Lane and E. Murphy, 1972). While it could be argued that this increased susceptibility is caused by a closely linked susceptbility locus, the simplest hypothesis is that the situation is analogous to the Chediak-Higashi syndrome and that beige itself is the major susceptibility factor ( Lane and E. Murphy, 1972). 10

More recently it has been shown that the beige mouse also resembles the Chediak-Higashi syndrome of humans because in both situations granulocytes have decreased bactericidal activity ( Gallin et al., 1974). It has likewise been shown in both bgJ/bgJ ( Brandt et al., 1975) and bg/bg ( Brandt and Swank, 1976) mice that there is a correlation between abnormal lysosome structure and defective lysosome function. Thus beige mice secrete much less than normal amounts of lysosomal enzymes from the proximal tubule cells and, as a result, have increased lysosomal enzyme activity in their kidneys ( Brandt and Swank, 1976). Indeed, this finding has led Brandt and Swank to suggest that beige mice "are defective in intracellular motility of lysosomes and/or their fusion with cellular membranes." 11

Finally, while there is some evidence that all of the effects of the beige mutation are a consequence of some disturbance in lipid metabolism ( C. Oliver et al., 1976), whether this is so, and how it specifically relates to the pigmentary disturbance, remains to be elucidated. 12

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