Chapter 3

The b-Locus and c (Albino) Series of Alleles

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I. The b-Locus

A. Influence of B/B vs b/b Substitution on Coat Color

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

Four mutations have been assigned to the b-locus (or to one very closely associated with it) on the fourth chromosome of the mouse. B, the wild type allele at this locus, produces black ( Plate 1-E) and the most recessive allele, b, produces brown ( Plate 1-F) eumelanin. Thus b, when homozygous, changes the gray color of the wild mouse to a brownish hue, which is generally known as cinnamon agouti or cinnamon. This phenotype results from the occurrence of yellow banded brown (instead of black) hairs.

Inasmuch as b-locus alleles have no demonstrable influence on phaeomelanin synthesis, black and brown yellow mice are usually distinguishable only by their eye color (or by whether their extra-follicular melanocyte population is "brown" or "black"). However, because yellow mice often display variable amounts of sootiness as a consequence of the cumulative effect of "umbrous" genes, which promote the synthesis of eumelanin in the hairs of the dorsum (see Chapter 2, Section III), some Ay/—;B/— and Ay/—;b/b animals can be distinguished by coat color, especially if B and b are known to be segregating.

B. Other Alleles and Dominance Relationships

For the b series:
Gene (MGI) All Alleles (MGI)

The three other genes of the b-series are light ( Blt), cordovan ( bc), and white-based brown ( Bw). Whereas Blt and Bw appear to be dominant to B, i.e., the phenotypes associated with each of these genes are distinguishable when the allele is heterozygous with B, bc is recessive to black and dominant over brown. 1 these dominance-recessive relationships are not complete however. For example, Blt and Bw yield different phenotypes when homozygous and when heterozygous, with B or b, and cordovan ( bc/—) mice are indistinguishable from brown ( b/b) animals in the presence of dilute ( d/d) ( D.S. Miller and Potas, 1955). In fact, there is evidence that b can sometimes express itself when heterozygous with B. Thus Dunn and Thigpen ( 1930) found that the effect of the silver gene, si (see Chapter 6, Section II), is greatly intensified in B/b black mice, and Durham ( 1911) and Snell ( 1931) observed that a/a;B/b;p/p mice are a shade lighter than a/a;B/B;p/p animals (but see Little, 1913).

C. Influence of B/B vs b/b Substitution on Pigment Granules

The predominant effect of substituting b/b for B/B is to change the nature of the eumelanin from a black, fuscous black, or fuscous pigment to a carob brown or mummy brown pigment ( E. Russell, 1949b). This change in the color of the pigment granule is accompanied by a change in its size and shape. Brown granules are significantly smaller than black granules and display much less variation in size (E. Russell, 1949a, 1949b). 2 Whereas the granules found in the septules of black-pigmented hairs are usually either long-oval or oval in shape, the granules of brown-pigmented hairs are round (see Figure 4-6) ( E. Russell, 1949a). Because of these differences it has been judged that intensely pigmented brown phenotypes have only slightly more than one-third the volume of pigment of intensely pigmented black mice ( E. Russell, 1948). Nevertheless this is still about twice as much pigment as occurs in intensely pigmented yellow hairs.

D. Effects of b-Locus Alleles on Tyrosinase Activity

The effect of genic substitution at the b-locus on tyrosinase activity has been well investigated ( L. Russell and W. Russell, 1948; Foster, 1951, 1959; Fitzpatrick and Kukita, 1959; Coleman, 1962) and, contrary to what one might expect, brown mice have at least as much tyrosinase as black mice. Indeed, in most of these studies brown mice had twice the amount of tyrosinase as black animals. The study of Coleman ( 1962) is particularly pertinent inasmuch as he measured the incorporation of 14C-labeled tyrosine into a/a;B/b, a/a;Blt/Blt, a/a;bc/bc, and a/a;b/b skin. Whereas brown skin incorporated about twice as much tyrosine as black ( B/B or B/b) skin and light skin, cordovan skin was intermediate between black and brown in its uptake ( Table 3-1). Moreover, to rule out the possibility that these paradoxical findings were a consequence of the in vitro assay employed (see Foster, 1959) Coleman ( 1962) measured the uptake of tyrosine when injected into 4-day-old b/b and B/B mice and obtained similar results. This in vivo study also indicated that brown granules develop as rapidly as black granules so that the increased activity of b/b skin cannot be attributed to the fact that brown granules develop more slowly and hence retain more active tyrosinase sites on their surface ( Fitzpatrick and Kukita, 1959).

E. Site of b-Locus Activity

To pinpoint the stage in the development of the melanosome when the b-locus is believed to operate requires a brief description of the "normal" development of this organelle. According to Moyer ( 1961, 1963, 1966 melanosome development is initiated when thin "unit fibers," often contiguous with polysomes, aggregate to form "compound fibers" within a membranous boundary. As these fibers cross-link and become oriented parallel to each other the shape of the melanosome 3 becomes apparent and melanin is deposited at definite sites along the fibers of its matrix. 4 This deposition of melanin continues so that ultimately the details of the matrix of the melanosome are obscured by the electron dense pigment. When melanin synthesis ceases the melanosome represents a typical mature pigment granule. 5

Observations of B/— and b/b granules indicate that it is during that stage of melanosome development when melanin is deposited at discrete sites along the fibers of its matrix that the b-locus operates. Thus Moyer ( 1961) observed that when melanin was just beginning to be deposited, the second-order periodicity in black granules was longer (about 200 A) than the second-order periodicity of brown granules (about 113 A). If this second-order periodicity represents the active site of tyrosinase activity, the shorter distance between sites in brown granules would allow for a greater number of sites for this activity and, hence, for the greater overall tyrosinase activity of b/b skin. Thus, according to this scheme, subunits of the protein controlled by the b-locus are possibly involved in the formation of the parallel fibers of the melanosome which bind tyrosinase in a certain fixed ratio ( Wolfe and Coleman, 1966). 6

The b-locus also influences the size and shape of the pigment granule as well as the final molecular structure of the melanin deposited. When viewed under the electron microscope, the melanin in mature brown granules is flocculent and coarsely granular, whereas that of black granules is very finely granular, almost appearing homogeneous ( Moyer, 1966) (see Figure 3-1). 7 It therefore appears that the protein produced by the b-locus not only must provide a structural framework for the attachment of tyrosinase but also must somehow affect its activity.

F. Other Ultrastructural Observations

Besides the observations of Moyer ( 1961, 1963, 1966), Lutzner and Lowrie ( 1972), and Hearing et al. ( 1973) (see note 5), the ultrastructure of black and brown melanosomes has also been examined by Rittenhouse ( 1968a). She observed that sections of black hair bulb melanocytes contain a mixture of round and elongated melanized bodies suggesting a population of granules round in cross-section and oval in longitudinal section. On the other hand, the granules found in intensely pigmented brown hair bulbs were usually nearly spherical. Rittenhouse also noted that maltese dilution ( d/d) (see Chapter 4, Section I) influenced the morphology of b/b granules; a/a;b/b;d/d granules, like B/— granules, were usually oval in longitudinal section and round in cross-section. Moreover, she observed that whereas the unmelanized B/— granule consists of one or more rolled membranes, the internal framework of b/b granules resemble a tangled ball of strands. Although this last observation contrasts with those of Moyer ( 1963, 1966) who could not find any disruption of the pattern of granule framework in b/b mice, it should be noted that most of his preparations were derived from the pigmented epithelium of the eye rather than from the hair bulb.

In an attempt to integrate the mode of action of b-locus alleles, Foster ( 1965) has speculated that this locus is the structural gene for a protein the alteration of which can greatly influence the properties of the melanosome.

G. Light (Blt)

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

The Blt mutation, first described by MacDowell ( 1950) and extensively studied by Quevedo and his associates ( Quevedo and Chase, 1958; McGrath and Quevedo, 1965; S. Sweet and Quevedo, 1968) and Pierro ( 1963a), is either an allele of the b-locus, or is closely linked and epistatic to this locus. Homozygous Blt mice ( Plate 1-H and Figure 3-2) are phenotypically distinct from heterozygous Blt animals ( Figure 3-2). Whereas Blt homozygotes (known as "lights") have almost white fur, except for the hair tips which are "hair brown," Blt heterozygotes (known as "darks") display darker hair tips (chaetura drab in color) with pigment extending further down the shaft ( Grüneberg, 1952; Quevedo and Chase, 1958). The ventral hairs of both homozygotes and heterozygotes are substantially less pigmented than dorsal hairs, and with advancing age the coat color of both lights and darks become progressively lighter. Ay/a;Blt/B mice are indistinguishable from non-Blt yellow animals.

In agouti animals, the yellow subterminal band conceals the color of the hair tip in both lights and darks so that the influence of the Blt gene cannot be recognized until the hair is well grown. The distinction between Blt heterozygotes and homozygotes is also less conspicuous in agouti than in nonagouti mice ( Grüneberg, 1952).

Although darks of the same age heterozygous for B or b are essentially identical in appearance ( Quevedo and Chase, 1958), the number of uveal melanocytes is lower in Blt/b genotypes than in Blt/B ( Pierro, 1963a).

One of the most interesting coat-color interactions occurs between Blt/— and P/p. Whereas P usually is dominant to p (pink-eyed dilute) this is not the case in Blt/— genotypes (nor is it the case in b/b mice — see Chapter 4, Section II, B). The hairs of light and dark mice either homozygous or heterozygous for p do not display an absence of pigment in the lower section of their hair shafts ( Quevedo and Chase, 1958). Thus the uniform dark sepia coat color of Blt/—;P/p is considerably darker than the color of Blt/—;P/P animals. Blt/—;p/p mice are a light metallic grey ( McGrath and Quevedo, 1965).

The specific effects of the Blt mutation on pigment deposition have been described in detail by Quevedo and Chase ( 1958). Cleared hairs of a/a;Blt/Blt mice display large clumps of pigment granules which occur predominantly in the medulla of the shaft ( Figure 3-3a). The degree of pigmentation is variable from hair to hair but almost invariably there is a reduced number of pigment granules as one proceeds from the tip of the hair to its base. Indeed, the bottom half of many hairs are completely devoid of pigment and when it does occur most of it is contained in sporadic clumps, separated from each other by empty septa ( Figure 3-3b). The clumps of pigment are highly variable in size; some are large enough to disrupt several septa. Hairs from Blt heterozygotes present a similar picture except that they possess on the average more pigment per hair than Blt homozygotes. The clumps of granules also are fewer and appear smaller in heterozygotes.

As far as the individual pigment granules are concerned, they are essentially identical in Blt/Blt, Blt/B, and Blt/b genotypes and possess similarities with both B/B and b/b granules. Their color, although lighter than those of B/B mice, is definitely of the black, rather than the brown, species. On the other hand, although the size and shape of the granules vary, most of them are round and closer to the size for brown than they are to the size for black ( Quevedo and Chase, 1958).

As might be expected from the phenotype, a/a;Blt/—;P/p animals possess pigment throughout the length of their hairs ( Figure 3-3c). These animals are also deficient in the large clumps of pigment typical of Blt/—;P/P animals although some small clumps occasionally occur. 8

The behavior of the melanocytes in the hair bulbs of Blt/— genotypes, as well as the pigment they contain, are a direct reflection of the situation in the hair shaft. Thus the pigment granules within the follicular melanocytes of a/a;Blt/Blt, a/a;Blt/B, and a/a;Blt/b mice, like those in the hair shaft, resemble black granules in intensity and brown granules in morphology. Blt/— melanocytes appear to contain fewer granules than B/B melanocytes and some of this pigment occurs in clumps, similar in size to those observed in the hair shaft ( Figure 3-4). This suggests either that large portions of Blt/— melanocytes or entire melanocytes are incorporated into the growing hair ( Chase and Mann, 1960). Consistent with this conclusion is the fact that many hair bulbs lack melanocytes completely or possess only one or two of these cells during the latter half of hair growth ( Figure 3-5). Indeed, 2 weeks after a new hair growth cycle is induced in light mice by the plucking of resting hairs, very few follicles contain active melanocytes, and some of these resemble the amelanotic melanocytes of albinos ( Figure 3-6). By the termination of hair growth, many of the hair follicles of light mice resemble those in "white-spotted" areas in that no trace of any melanocytes can be found ( Figure 3-7) ( Chase, 1958). The situation in Blt heterozygotes is similar except that active melanocytes persist in more hair follicles over a greater course of the cycle ( Quevedo and Chase, 1958).

In accord with the situation in the hair shaft of Blt/— mice heterozygous ( P/p) or homozygous for p, the melanocytes of the hair bulb persist throughout the growth cycle, apparently releasing pigment granules to epithelial cells at a normal rate. Moreover, the melanocytes of these genotypes remain dendritic and have not been observed to be incorporated into the hair shaft ( McGrath and Quevedo, 1965; S. Sweet and Quevedo, 1968).

Although all the melanocytes in the hair bulb of light mice are clearly dendritic during early stages of the hair growth cycle, some rounded, densely melanized cells deficient in dendrites (known as "nucleopetal" cells — see Chapter 4, Section I, D) are subsequently found in the upper part of the follicle ( Figure 3-8). The frequency of these cells increases so that the uprooted cells (or portions thereof) which become incorporated into the developing hair shaft also usually lack dendrites, are densely melanized, and may contain pycnotic nuclei ( S. Sweet and Quevedo, 1968). Indeed, as will be reiterated when the effects of the d and ln loci are discussed ( Chapter 4, Section I, D), the very fact that nucleopetal melanocytes develop in Blt hair follicles may be responsible for their being uprooted and incorporated into the hair, i.e., a well-developed dendritic system may serve as a mechanical anchor. Why these cells develop, however, is not known. One possibility is that the Blt gene causes follicular melanocytes to produce pigment granules at a faster rate than they can deliver them to keratinocytes and as a consequence the melanocytes enlarge, become detached from their normal matrix positions, and are swept into the hair shaft ( Quevedo and Chase, 1958; S. Sweet and Quevedo, 1968). 9 If this mechanism is correct one would expect the rate of melanin synthesis to be reduced in Blt genotypes heterozygous for p (since these animals do not display either nucleopetal melanocytes in their follicles or large clumps of pigment in their hairs). Unfortunately, concrete evidence that this is the case is lacking. Nevertheless, Blt/—;p/p as well as yellow-Blt mice almost certainly have a slower rate of pigment synthesis than Blt/—P/P mice and these genotypes also display melanocytes of normal morphology.

1. Interaction With Leaden and Dilute

Since the recessive mutations leaden ( ln) and dilute ( d) (see Chapter 4, Section I) also affect melanocyte morphology by transforming dendritic (nucleofugal) cells into cells with a poorly developed dendritic system (nucleopetal), some mention of how these genes interact with Blt, as well as a comparison of the effect each of these genes has when combined with other coat-color determinants, is germane. Indeed, the results of these interactions demonstrate clearly that the influence Blt has on melanocyte morphology is quite distinct from that of d or ln ( McGrath and Quevedo, 1965; S. Sweet and Quevedo, 1968).

Inasmuch as d/d hair bulb melanocytes are always nucleopetal, one might anticipate that when d/d is combined with Blt/— these nucleopetal cells would aggravate the "diluting" action of Blt by accelerating the rate at which melanocytes become incorporated into the hair shaft. This, however, is not the case. Although in a/a;d/d;Blt/— mice the morphology of the follicular melanocytes are consistent with their d/d constitution, they do not appear to be lost from the hair bulbs more frequently than those of nondilute light or dark nonagouti mice. Indeed the dynamics of melanocyte behavior in dilute-Blt mice seems to be more comparable to those of Blt heterozygotes than homozygotes ( McGrath and Quevedo, 1965). This observation is important because it indicates that factors other than melanocyte shape are undoubtedly involved in determining whether melanocytes are dislodged from the hair bulb and incorporated into the septules of the hair ( McGrath and Quevedo, 1965).

Among the observations which indicate the Blt differs from ln and d in the manner in which it influences melanocyte morphology are the following: (1) Whereas Blt has no influence on the morphology of yellow melanocytes, both d/d and ln/ln yellow mice display nucleopetal cells (Poole and Silvers, unpublished); (2) p, even when heterozygous, can influence the morphology and behavior of Blt/— melanocytes, whereas this is not the case even when p is homozygous in combination with either ln/ln ( S. Sweet and Quevedo, 1968) or d/d (Poole and Silvers, unpublished); and (3) while the hairs of Blt genotypes, particularly homozygotes, become unpigmented prematurely as a consequence of the fact that the melanocytes which are incorporated into the hair are not replaced, this is not the case in ln/ln and d/d animals. Thus, although the melanocytes of leaden and dilute mice are also incorporated periodically into the hair, they appear to be replaced so that a population of follicular melanocytes persists to pigment the entire shaft.

Finally it should be noted that in contrast to the situation in ln/ln and d/d genotypes and, indeed, in contradistinction to the situation in Blt hair bulbs, the extrafollicular melanocytes of light mice are usually nucleofugal in morphology. Thus, light melanocytes in the dermis and epidermis of the sole of the foot, ear pinna, scrotum, and tail ( S. Sweet and Quevedo, 1968; Quevedo, 1969b), as well as those in the harderian gland ( Markert and Silvers, 1956), all display well-developed dendrites ( Figure 3-9). Why this is the case is not known but one possibility, which will be discussed further when we concern ourselves with the action of genes at the d and ln loci ( Chapter 4, Section I, E), is that light melanocytes have an intrinsically greater capacity to develop well-melanized dendrites than either leaden or dilute melanocytes ( Quevedo, 1969b). In the compact environment of a hair bulb light, leaden and dilute melanocytes are unable to extend their dendrites whereas in less compact extrafollicular locations, light melanocytes, but not dilute or leaden cells, develop normally.

2. Can a Melanocyte Pigment More Than One Hair?

The observation that by the end of a given hair cycle, but when the hair is still growing, the hair bulbs of lights and darks appear to be devoid of melanocytes, and are morphologically similar to the nonpigmented bulbs of white-spotted mice which are known to lack pigment cells ( Chase and Rauch, 1950; Silvers, 1956), is important because it relates to the question of whether melanocytes (or their mitotic descendents) can pigment more than one hair. Thus if the disappearance of mature melanocytes from the hair follicles of Blt mice is due to all of them being incorporated into the shaft, the melanocytes for the next hair generation would have to be derived either from some unpigmented stem cells in the epithelium, or from cells in the dermal papilla (Chase, 1951, 1958) as is the case for feathers ( Foulks, 1943). On the other hand, it is also possible that at least some melanocytes revert to a dormant form which is indistinguishable from the epithelial cells of the follicle and are reactivated, perhaps producing mitotic descendents, when the next hair cycle is initiated ( Chase, 1958; Quevedo and Chase, 1958). Although according to Chase ( 1958) the bulk of evidence supports the contention that there are stem cells at definite sites of the hair follicle which give rise to the mature and expendable melanocytes (see also discussion following Chase and Mann, 1960), and the situation in Blt follicles is consistent with this, there is also evidence (see Chapter 11, note 17) which indicates strongly that melanocytes (or their mitotic descendents) can, in fact, pigment more than one hair. Perhaps both possibilities occur, i.e., while some melanocytes may be able to pigment more than one hair, a stem cell source of new cells may also operate as an insurance mechanism.

3. Influence on the Pigmentation of the Eye

Although Blt heterozygotes and homozygotes have black eyes, the substitution of Blt for B does result in a decrease in the amount of eye pigment, a decrease which is more pronounced in homozygotes than in heterozygotes ( Pierro, 1963a). While this decrease is caused predominantly by a reduction in the number of melanocytes in the uveal tract, the number of pigment cells is reduced in the choroid of both lights and darks, and probably in the iris region of lights, as well. Nevertheless, the amount of pigment in the eyes of Blt homozygotes and heterozygotes is somewhat greater than in b/b eyes ( Pierro, 1963a).

H. White-Based Brown ( Bw)

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

The last member of the b-series, white based brown ( Bw), occurred following the exposure of spermatogonia to 600 r low-dose rate gamma-irradiation ( Hunsicker, 1969). This mutation resembles Blt in some of its effects and, like Blt, the possibility that it is closely linked to B and b has not yet been ruled out completely. As in the case of Blt, Bw mice display a reduction in the amount of pigment at the base of the hair which is more pronounced in homozygotes than in heterozygotes. However, in contrast to the situation in Blt genotypes (where animals heterozygous for B or b are phenotypically identical), Bw/B mice are clearly different from Bw/b animals. In Bw/B animals only the extreme base of the hair is light, the remainder being just barely distinguishable from B/B dorsally, though clearly lighter ventrally. On the other hand, one-quarter to one-third of Bw/b dorsal hairs are near-white proximally, and the portion which is pigmented is phenotypically brown. Moreover, whereas the eyes of Bw/B mice are full colored, those of Bw/b animals appear brown. Bw homozygotes resemble Bw/b heterozygotes except that the proximal, near-white portion of the hair is about twice as wide ( Hunsicker, 1969).

I. Influence of b-Locus on Multiple Forms of Tyrosinase

The tyrosinase of mice, like that in other organisms, has been shown to occur in a number of forms separable by acrylamide-gel electrophoresis ( Wolfe and Coleman, 1966; Burnett and Seiler, 1966; Burnett et al., 1967, 1969; Holstein et al., 1967, 1971; Burnett, 1971; Quevedo, 1971). Although B, Blt, and bc have no significant effect on the typical tyrosinase pattern consisting of three uniformly darkened bands (T1, T2, and T3) of dopa melanin, this is not the case for the b allele. a/a;b/b animals display normal darkening of T1, but considerably less dopa melanin is deposited in the T2 and T3 bands ( Holstein et al., 1967; Quevedo, 1971). The significance of this difference is not known.

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