Chapter 2

The Agouti and Extension series of Alleles, Umbrous and Sable

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II. The Extension Series of Alleles

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

It has been known for some time that there are a number of mammals (e.g., rabbit, guinea pig, dog) which have a series of alleles that extend or restrict the amount of eumelanin, with an opposite effect on phaeomelanin (See Searle, 1968b). It is therefore not surprising that the mouse has a comparable series. What is surprising, however, is that this has only relatively recently been established. Thus, while as long ago as 1912 Hagedoorn reported a recessive mutation that caused yellow coat color, because the strain carrying this mutant soon became extinct, it was not until sombre was described in 1961, and recessive yellow was reported again in 1968, that an "extension series of alleles" for the mouse finally was confirmed. This series of alleles is now known to be located on chromosome 8 ( Falconer and Isaacson, 1962).

A. Sombre (Eso)

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

1. Origin and Influence on Pigmentation

This dominant mutation 26 has an interesting history in that it appeared within 3 days in two unrelated C3H litters, in each case from dams that had been drenched with dibutylphthalate, an antiectoparasite agent. Indeed, because of this situation it was initially suspected that these deviants represented "phenocopies" and not true mutations ( N. Bateman, 1961).

Aside from the fact that on the C3H ( A/A) background on which Eso occurred Eso/E heterozygotes have dark ears and mammae, they resemble nonagouti ( a/a) mice. Often when nonagoutis are mature their flanks are flecked with yellowtipped hairs and their ventrums are grey. These same features occur in A/A;Eso/E mice where, according to Bateman, they are even more conspicuous. Moreover, both a/a;E/E and A/A;Eso/E mice have considerable numbers of "yellowed" perineal hairs right from their first pelage.

A/A;Eso/Eso mice are strikingly black ( Plate 1-G) and are easily recognized from Eso heterozygotes from as early as 12 days of age. However, at least on the C3H background, they fall short of being completely black, as they display a few "yellowed hairs" on the perineum. In fact, as Bateman notes, it is only this feature which spoils their perfect mimicry of extreme nonagouti ( ae/ae). Viability and fertility are normal in both heterozygotes and homozygotes.

2. Interaction with Other Coat Color Determinants

Insofar as the interaction of Eso with other mutants is concerned, Bateman remarks that Aw/A;Eso/E heterozygotes, unlike Aw/A;E/E animals, do not have light bellies; presumably they are indistinguishable from A/A;Eso/E genotypes.

G. Wolff and his colleagues ( 1978) have compared viable sombre ( Avy/A;Eso/E) with agouti sombre ( A/ae;Eso/E) mice and although they found both of these genotypes to be black 27 they nevertheless could usually be distinguished on the basis of body weight and ventral pigmentation. Sombre Avy males reached a mean body weight of 44.2 +/- 0.6 g at 18 weeks, whereas sombre A males had a mean weight of 30.7 +/- 0.4 g at the same age. It thus appears that Eso is epistatic to Avy only with regard to coat color. 28 This is important because it indicates that the abnormally regulated synthesis and/or deposition of fat caused by Avy (and Ay) is in no way related to phaeomelanin synthesis ( G. Wolff et al., 1978).

With regard to the ventral pigmentation of Avy/A;Eso/E and A/ae;Eso/E animals Wolff and his associates observed that, in general, it was more dilute in mice of the former genotype. Microscopic examination of plucked hair samples showed that the most noticeable difference occurred in the monotrichs. In agouti sombre mice these hairs are heavily and uniformly pigmented throughout their length, whereas those of viable yellow sombre animals are not heavily pigmented at the tip and tend to display over their entire length a somewhat uneven distribution of eumelanin granules. The bases of these monotrichs also frequently lack pigmentation. 29

The coat of both viable yellow sombre and agouti sombre mice appear to possess a faint yellow cast in certain regions of the body. These include the ventral neck region, the chest, the femoral region of the forelimb, the base of the ear, and the perianal region. Nevertheless, microscopic examination of plucked hairs from these areas failed to reveal any phaeomelanin granules, the hairs being either devoid of pigment or dilute black. Since hairs from these various regions are much finer than those of the main pelage, Wolff and his associates believe that this property, in combination with their dilutely pigmented (or unpigmented) condition, is responsible for their faint yellow appearance.

One of the most interesting observations in this study was that many, but not all, zigzags from agouti sombre (and probably to a lesser extent from viable yellow sombre) mice lack pigment or contain relatively little in exactly the same region which is pigmented yellow in A/—;E/E animals, i.e., in the apical region of the shaft which precedes the first constriction. Consequently, although no yellow pigment is detected in these mice, the sparse pigmentation which takes its place yields a pattern similar to the agouti pattern. 30 This "banding" pattern is important because it indicates that eumelanin synthesis is inhibited at definite periods during hair growth regardless of whether phaeomelanin synthesis occurs or not ( G. Wolff et al., 1978).

B. Tobacco Darkening (Etob)

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

1. Origin and Influence on Coat Color

This allele of the extension series received its name from the fact that it originated in the tobacco-mouse (Mus poschiavinus Fatio), 31 where it is responsible for the darkening of the coat. According to von Lehmann (1973), as a consequence of this gene young ( A/A;Etob/Etob) tobacco-mice have a nonagouti back and "cannot be distinguished from black mice until the 8th week; later the flanks become agouti." When tobacco mice are crossed with nonagouti black ( a/a;B/B) house mice the F1 ( A/a;Etob/E) display a wild type pelage but the dorsum may be darkened. Crosses to yellow ( Ay) or agouti (A) house mice produce yellow or agouti mice with darkened backs, though less so with agouti. The expression of Etob in subsequent brother x sister generations derived from the (house x tobacco mouse)F1 indicates that, when homozygous, Etob is epistatic over nonagouti. It therefore appears that Etob, like Eso, is incompletely dominant over E. This is substantiated further by the observation that when A/a;Etob/E animals are backcrossed to tobacco mice ( A/A;Etob/Etob) half of the offspring are dark ( A/—;Etob/Etob) and half are grey with some darkening of the back ( A/—;Etob/E) ( von Lehman, 1973). In a subsequent report von Lehman ( 1974) presents evidence that tobacco darkening is a member of the E-series. 32

C. Recessive Yellow (e)

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

1. Origin and Influence on Coat Color

This mutation occurred in Hauschka's C57BL/6 subline and is very likely a recurrence of Hagedoorn's mutant. Although the coat of adult e/e mice is a clear yellow and, according to Hauschka and his associates ( 1968), somewhat less orange than the coat of Ay/a;E/E animals of the YBR/Wi strain, prior to weaning it displays some "dorsally concentrated umbrous sootiness" which diminishes with successive molts. In fact, this sootiness is so intense that it is difficult to distinguish a/a;e/e from a/a;E/E when the hairs first emerge through the skin of infant animals (see Poole and Silvers, 1976b). 33 Like Ay/a mice, the eyes of a/a;e/e animals are black.

2. Recessive Yellow and Lethal Yellow: Similarities and Differences

Histological examination of the yellow granules in the coat of e/e mice revealed that they were much the same as the granules in Ay/— animals. Nevertheless, the cortex of e/e hairs appeared to contain significantly less pigment than the cortex of Ay/— hairs ( Hauschka et al., 1968). 34

In both e/e and Ay/a mice phaeomelanin can be extracted from the hairs by the same treatment; immersion in 10% potassium hydroxide for 15 minutes ( Hauschka et al., 1968). In both yellow genotypes all extrafollicular melanocytes also produce only eumelanin. Thus Lamoreux and Mayer ( 1975) observed that the melanocytes located in the dermis of the skin, leg muscles, choroid, harderian gland, and meninges of the brain and spinal cord of young e/e mice were all black. Moreover, although exact counts of melanocytes were not made, these investigators estimated that their numbers were similar in these locations in a/a;e/e and a/a;E/E genotypes. The melanocytes of e/e ear skin likewise possess eumelanin but here, as in Ay/— ear skin, they are not, for some unknown reason, as numerous as in a/a;E/E ear skin (Poole and Silvers, unpublished).

Among the numerous differences between recessive and lethal yellow is the fact that mice homozygous for the former are viable and, unlike Ay/a;E/E animals, breed well, do not become obese, and, at least on the basis of current evidence, are no more susceptible to tumors than a/a;E/— genotypes. Furthermore, while leaden ( ln/ln) has been reported to be epistatic to e/e ( Hauschka et al., 1968), this is not the case with Ay/—. (For other differences see notes 33 and 35; Section II, D; Chapter 3, Section II, C; Chapter 4, note 9; and Chapter 12, Section III.)

As with sombre it appears that while e/e is epistatic to Ay/— in terms of its effect on pigmentation ( Searle and Beechey, 1970), it probably does not interfere with other manifestations of the lethal yellow allele. Thus while the coats of Ay/a;e/e mice are identical with those of a/a;e/e animals they become as obese as Ay/a;E/— genotypes ( Lamoreux, 1973). 35

3. Site of Gene Action

Inasmuch as recessive yellow is the only coat-color determinant besides those of the agouti series which promotes the synthesis of phaeomelanin, it was important to determine its site of action. Does it act via the follicular milieu, as is the case for the agouti series of alleles, or does it act within the melanocyte? Two independent investigations have been carried out to answer this question and the results of both are in accord with the premise that it is the e/e genotype of the melanoblast which determines its phaeomelanin-synthesizing activity; nevertheless, these cells produce yellow pigment only in a hair follicle environment.

In the first of these studies ( Lamoreux and Mayer, 1975) grafts composed of 9-day-old neural tube, including neural crest cell, and 11-day-old neural crest-free embryonic skin were placed in the coelom of the chick and allowed to differentiate for 15 days. It was found that when a/a;e/e neural tube was grafted with a/a;E/E skin, the hairs of recovered grafts were pigmented yellow, whereas a/a;e/e skin produced black hairs when combined with a/a;E/E neural tube.

In a simultaneously conducted series of experiments, Poole and Silvers ( 1976b) determined the kind of pigment produced by various dermal-epidermal combinations of e/e and E/E embryonic skin when allowed to differentiate in the testes of adult hosts for 3 weeks. They found that when 13- to 15-day-old a/a;E/E epidermis (which possesses numerous melanoblasts) was combined with a/a;e/e dermis (which is either melanoblast free or contains very few melanoblasts), that the hairs which formed were completely black. On the other hand, in the reciprocal combination, consisting of black ( a/a;E/E) dermis and recessive yellow ( a/a;e/e) epidermis, all the hairs were pigmented in the recessive yellow pattern, i.e., they were composed predominantly of phaeomelanin with eumelanin at the tips. Poole and Silvers also determined the behavior of dermal-epidermal recombinants that differed at both the extension and yellow loci. This was accomplished by recombining recessive yellow ( a/a;e/e) and agouti ( A/A;E/E) dermis and epidermis. They found that when a/a;e/e dermis was combined with A/A;E/E epidermis all the hairs which developed were black, i.e., the A/A;E/E melanoblasts in the epidermis had responded to the a/a dermis. However, the reciprocal combination of A/A;E/E dermis and a/a;e/e epidermis developed hairs pigmented with the recessive yellow pattern (since in this case the hairs were pigmented by a/a;e/e melanocytes).

Since a/a;e/e melanocytes did not respond to any influence from either an a/a or A/A dermis, a final series of experiments was aimed at introducing these cells into nonagouti black (but nonpigmented) hair follicles. This was accomplished by combining 12-day-old embryonic recessive yellow dermis (which still possessed a population of melanoblasts) with a/a;c/c (albino) epidermis of the same age. Although, as a consequence of the fact that this epidermis too possessed melanoblasts, not all the hairs which developed in these grafts were pigmented, i.e., some were populated only by amelanotic melanocytes (see Chapter 10, Section I, D), nevertheless, those hairs which were pigmented all displayed the recessive yellow pattern. the results of these various dermal-epidermal recombinations are summarized in Table 2-6.

It therefore seems evident that even though e/e melanocytes produce yellow pigment only in hair follicles, recessive yellow, unlike the alleles at the agouti locus, acts autonomously within the melanoblast. What it is that is unique about the follicular environment which turns on phaeomelanin synthesis in e/e pigment cells is not known, but perhaps it is related to the fact that melanocytes in hair follicles synthesize melanosomes at a much more rapid rate, for inclusion into the growing hair, than extra-follicular cells ( Lamoreux and Mayer, 1975).

D. The Effect of Melanocyte-Stimulating Hormone (MSH) on Phaeomelanin Synthesis

Geschwind and his colleagues ( Geschwind, 1966; Geschwind and Huseby, 1966, 1972; Geschwind et al., 1972) have studied the effect of alpha-MSH on the coat color of various genotypes. These efforts stemmed from the observation that the coat of an agouti mouse which had been maintained on stilbestrol for 11 months, and which had developed a pituitary tumor, was much darker than normal. Transplantation of the tumor to other agouti mice also maintained on the same stilbestrol-containing diet likewise produced darkening of their coats and microscopic examination of dorsal hairs from these mice revealed that the yellow band had been obliterated completely. It was subsequently found that the injection of alpha-MSH into otherwise untreated agouti mice produced the same effect and attempts were made to modify the phenotypes of other genotypes either directly by raising circulating MSH levels with injections of the hormone or indirectly by transplanting the tumor. However, these treatments only influenced the expression of the agouti locus where the most obvious effect was to convert the pigmentation to the nonagouti type. Thus in a/a animals homozygous for either brown ( b), albinism ( c), dilute ( d), or pink-eye ( p), MSH had no effect. 36 Its most dramatic influence was on the coat of the Ay/— mouse; in regions which had been plucked 5-8 days previously or which were in active growth at the time of hormone treatment, only intensely black or brown colored hairs, in Ay/—;B/— and Ay/—;b/b genotypes, respectively, emerged (see Figures 2-5 and 2-6). 37 This influence of the hormone on phaeomelanin was also recognizable in Ay/—;p/p animals where normally the hairs are basically grey with subterminal yellow bands which gives an overall yellow appearance to the coat. Following the administration of MSH, however, growing hairs were totally grey and the coat was indistinguishable from a/a;p/p genotypes.

In spite of the fact that MSH converted phaeomelanin to eumelanin synthesis when the former resulted from the action of agouti locus alleles, this was not the case when the yellow pigment was a manifestation of recessive yellow ( e/e). Thus the color of the recessive yellow mice was completely unaltered either by MSH administration ( Figure 2-5 ) or by the presence of the tumor. Recessive yellow mice heterozygous for lethal yellow, i.e., Ay/a;e/e, also continued to produce phaeomelanin when exposed to MSH. This provides further evidence that the pigmentation of these genotypes is identical with that of a/a;e/e animals. Moreover, in contrast to the site of action of agouti locus alleles, since the E-locus acts within the melanoblast, it also indicates that the influence MSH has on converting phaeomelanin synthesis to eumelanin production is mediated via the follicular milieu. 38

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