Notes to Chapter 3

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1Cordovan received its name from the fact that it produces a phenotype which resembles the rich brown of cordovan leather. The mutation was recognized in an F1 litter from a cross of a C57BL/10 male by a DBA/1 female. Hence the mutation must have originated in the C57BL/10 stock. The same male which sired the litter containing the mutant sired six other F1 litters (five by the same female), in none of which cordovan appeared ( D.S. Miller and Potas, 1955).

2According to Moyer ( 1966) in melanocytes of neural crest origin brown granules average about 0.5 x 0.8 microns whereas black granules average about 0.5 x 1.5 microns.

3There have been some discrepancies in the utilization of the terms "premelanosome," "melanosome," and "melanin granule." As originally proposed ( Seiji et al., 1961) the term premelanosome was defined as a distinctive particulate protein matrix upon which melanin was usually deposited with consequent formation of the melanosome. Thus the melanosome represented a premelanosome after the onset, but prior to the completion, of melanin synthesis which characteristically possessed an active tyrosinase system. The melanin granule, on the other hand, represented a melanin-containing organelle in which melanization was complete and no tyrosinase activity could be detected. However, because certain difficulties arose in the utilization of these terms (see Fitzpatrick et al., 1967) in 1967 Fitzpatrick and his associates proposed new definitions. They requested that the term melanosome be employed to designate the fully pigmented melanin-containing organelle only. The premelanosome would then be used to designate all stages in the genesis of melanosomes that precede the fully developed state. Finally, they suggest that the term melanin granule be used to include all melanin-containing particulates that can be observed with light microscopy.

4According to Moyer ( 1966), "inspection of the fibers in melanin granules at high magnifications reveals further structural details. The unit fiber is approximately 35 Å in diameter and generally appears as a loosely coiled helix with a variable pitch of several hundred Ångström units. The compound fibers are approximately 90 Å in diameter. In longitudinal section they appear to be hollow cylinders with a lumen approximately 35 Å in diameter and walls approximately 35 Å thick. In cross section they do not appear to be hollow. The cross-links that form between compound fibers during their aggregation into matrix sheets are fairly uniform in size. They are approximately 100 Å long and 30 to 40 Å wide. Thus, the final matrix on which melanin is deposited appears to be a lattice of parallel fibers spaced approximately 100 Å apart and cross-linked by fibers approximately 35 Å in diameter. Cross sections of melanin granules show that this flat lattice-like sheet is rolled up like a rug to give the mature granule its three dimensional form."

5The ontogeny of a/a;B/B pigment granules has also been described by Lutzner and Lowrie ( 1972) and by Hearing et al. ( 1973). Hearing and his colleagues observed that premelanosomes in the retina and choroid of a/a;B/B mice are fibrillar in appearance ( Figure 3-15a), measure about 35 - 45 Å in diameter, and sometimes possess a helical or zigzag structure. "The axial diameter of the filaments is about 60 Å, and the pitch of the helices is between 150 and 200 Å." Occasionally a striated periodicity is visible, oriented perpendicular to the length of the filament, and is in the order of 95 - 100 Å. Moreover, sometimes "the filaments are arranged in phase so that the striated periodicity observed imparts a periodicity on the entire granule." Melanin is deposited rather uniformly on the filaments as the granule matures ( Figure 3-15b), and as this deposition builds up the filaments fuse ( Figure 3-15c). Frequently in mature granules, "circular electron lucent areas are observed, especially at the periphery of the granule; these measure 200-300 Å in diameter" ( Figure 3-15c). Lutzner and Lowrie's observations, which also were confined to granules of the eye (choroid, retina, and iris), are similar to those of Hearing et al. Their paper includes a schematic diagram of how they believe melanin formation proceeds and this diagram is reproduced here ( Figure 3-16) as a means of providing a concise summary of their views. In contrast to Moyer (see note 4; and it should be noted that Moyer's unit and compound fibers compare with Lutzner and Lowrie's melanofilaments and youngest melanotubules, respectively), these investigators suggest that "rather than cross-linking to form matrix sheets, young melanin tubules widen to 600 Å in diameter and then partially fuse." Moreover, they prefer the notion that melanin is deposited around each melanofilament and that the melanin itself forms the walls of the melanotubule, rather than being deposited on a matrix sheet of aggregated compound fibers.

6Since, as noted ( Table 3-1), tyrosinase activity in bc/bc skin is intermediate between that of B/— and b/b mice this hypothesis would be strengthened if it was found that the second-order periodicity of b/bc melanosomes was likewise intermediate between those of black and brown melanosomes. As far as I am aware this information is not available.

7Moyer ( 1966) also points out that as a result of the fact that black granules are finely granular and brown granules flocculent (see also Hearing et. al., 1973), black granules appear much denser than brown granules; they also tend to shatter more readily during sectioning than brown ones.

8The fact that clumping of eumelanotic pigment granules is significantly reduced in mice heterozygous for both Blt and p is surprising. Since some clumping is observed in pink-eyed non-Blt mice (see Chapter 4, Section II, D), more clumping might be anticipated in darks ( Blt/—) heterozygous for p than in P/P darks ( Quevedo and Chase, 1958).

9On the other hand, the possibility should not be overlooked that the primary effect of Blt is on the capacity of the melanocyte to extend dendrites. If such an effect influenced the rate at which these cells delivered granules to keratinocytes, this, in turn, might influence their stability in the bulb ( S. Sweet and Quevedo, 1968).

10Mayer and Oddis ( 1977) have isolated amelanotic melanocytes from cultures of embryonic albino epidermis. The isolation procedure was based upon the fact that while melanoblasts differentiate in vitro only when in contact with epidermal cells, once differentiated these cells persist even though the epidermal cells do not survive in culture for more than 11 days. When 14-day-old pigmented ( a/a;b/b) and albino ( c/c) embryonic epidermis, respectively, were cultured in vitro the pure populations of cells which persisted were identical in every respect save that the former were pigmented while the latter were not. On the other hand, no such cells were found when similar procedures were carried out on epidermal explants from white-spotted ( W/W and Sl/Sl) mutant embryos (see Chapter 7, Section VII).

11Detlefsen referred to this mutation as cd.

12In 1967 A.J. Bateman described an a/a;cch/c mouse which possessed two differently colored areas on each side of the dorsum just posterior to the midline of the trunk. One of these patches was a medium shape of sepia, i.e., it resembled the a/a;cch/cch phenotype; the other was white. To explain this mosaicism Bateman suggests that it resulted from somatic crossing-over between the c locus and the centromere in a primordial melanoblast of the neural crest, and that the complementary daughter cells ( cch/cch and c/c) lined up on opposite sides of the mid-dorsum (see Chapter 7 and Chapter 8, Section I). An alternative explanation for the twin spots would be nondisjunction of the cch-bearing chromosome, producing complementary clones which would be trisomic ( cch/cch/c) and monosomic ( c). However, because chromosome 7 is quite large and important, Bateman does not believe that this explanation is as likely since the viability of the monosomic line would be doubtful. Other possible examples of somatic crossing-over in the mouse include phenotypes described by Keeler ( 1931), Dunn ( 1934), and Carter ( 1952; see Chapter 10, note 7). For a detailed review on the case for somatic crossing-over in mice and other mammals see Grüneberg ( 1966a). For other mechanisms by which mosaicism may be produced see Robinson ( 1957), L. Russell ( 1964), and L. Russell and Woodiel ( 1966).

13 Ay/a;cp/cp mice bear a striking resemblance to Aw/Aw;cch/c;p/p animals, i.e., cch/c heterozygotes of strain 129.

14In 1939 Mohr described a mouse caught on the German island of Spiekerog, which resembled the Himalayan rabbit in its fur pigmentation. Although no breeding tests were carried out with this animal it is quite likely that its phenotype also resulted from a c-locus mutation to "himalayan." An independent mutation to ch has also been reported by Wallace ( 1972). It occurred in a cch/cch x cch/cch mating. According to Wallace a/a;ch/ch, unlike A/A;ch/ch, "are darker in the nest than as adults."

15All but one of these fractional mutants were fully viable when homozygous; a situation which contrasts with those for the d-locus where only 3 of 15 tested spontaneous mutation proved viable, most of the remainder being of the dop type (see Chapter 4, note 8) ( L. Russell, 1971).

16These lethal albino "alleles," which have been studied most extensively by Gluecksohn-Waelsch and her associates ( Erikson et al., 1968; Thorndike et al., 1973; Trigg and Glueckson-Waelsch, 1973; Erickson et al., 1974a, 1974b; Gluecksohn-Waelsch et al., 1974, 1975; D.A. Miller et al., 1974; Garland et al., 1976; S. Lewis at al., 1976), including the following: c6H and c25H, which are early embryonic lethals and c14CoS, c112K, c65K, and c3H which are lethal within a few hours after birth [nevertheless c3H/c6H compounds are viable but stunted and sterile, usually dying at approximately 2 months of age ( Searle, 1961)]. Studies of the latter four "alleles" have revealed ultrastructural abnormalities of the endoplasmic reticulum and Golgi apparatus in the liver and kidney of newborn homozygotes. These abnormalities are associated with deficiencies of at least three enzymes (glucos-6-phosphatase, tyrosine aminotransferase, and serine dehydratase) which in normal mice increase in the liver and kidney shortly after parturition. In addition, some serum proteins have been found to be significantly lower in these neonatal albino deviants. It has been suggested that the mutational effects observed in these animals result from deletions involving regulatory rather than structural genes at or near the c-locus ( Garland et al., 1976). Twenty four other lethal albino deviants known to represent deficiencies have been studied by L. Russell and Raymer ( 1979). These include 13 which are lethal before implantation, 10 shortly after implantation, and 1 neonatally.

17Microscopic examination of cleared hairs from Ay/a;C/c as well as from the ventrum of at/at;C/c mice has confirmed the visual reduction in phaeomelanin. In these hairs yellow granules are located in the distal portion of the shaft with virtually no granules in the proximal regions. This reduction in granule number is most striking in ventral hairs where in some instances, they are completely absent (Poole and Silvers, unpublished).

18 C/C and C/c genotypes have also been reported to be distinguishable by means of X-rays. Following low doses of irradiation Hance ( 1928) reported that the regenerating hairs of A/A;C/c mice were mostly white whereas most hairs of A/A;C/C animals tended to be even darker than those of untreated controls, with only a slight admixture of white hairs. This observation, however, remains to be confirmed ( Chase, 1949). In fact, extensive tests of C/C and C/c in combination with agouti and nonagouti have failed to indicate a difference in this "greying" response from X-irradiation (Chase, unpublished) and the only phenotype that does respond differently is silver ( Chase and Rauch, 1950) (see Chapter 6, Section II, G).

19It should also be noted that although a/a;b/b;cch/cch mice resemble a/a;b/b;C/C animals they incorporate only about one-fifth as much tyrosine, Moreover, the increased incorporation of tyrosine displayed by intensely pigmented brown over intensely pigmented black mice is not evident in the presence of cch/cch (see Table 3-1).

20Quevedo et al. ( 1967) have studied the influence of environmental temperature on UV-induced tanning of himalayan ( ch/ch) and intensely pigmented black (C57BL/St) mice maintained at 10, 25, and 34 C. They found that whereas in himalayan animals there was a significant activation of epidermal melanocytes in UV-irradiated plantar skin at 10 and 25 C but not at 34 C, no UV activation of epidermal melanocytes occurred in the depilated hairy skin at any of these temperatures. On the other hand, at all temperatures the irradiated plantar and hairy skin of C57BL/St mice displayed a striking increase in melanogenically active epidermal melanocytes as well as in heavily melanized malpighian cells. Quevedo and his associates have also shown that ch/ch and C/C skin grafts transplanted to (C57 x himalayan)F1 histocompatible hosts (as well as grafts exchanged between histocompatible Ay/A;B/B and A/A;B/B littermates) respond to UV as in situ. Thus the response of epidermal melanocytes to UV appears to be determined primarily by the genetic and physiologic constitution of the irradiated skin and not by more remote systematic factors ( Quevedo et al., 1967).

21The effects of various c-series alleles on tyrosine hydroxylase and melanin production has also been investigated at the University of Edinburgh ( Bulfield, 1974). In these studies cch/cch mice were found to produce an enzyme with lower Vmax and higher Km than C/C animals. c/c had less than 10% of C/C activity. It was not established whether this was "residual," unspecific, activity or allele-specific activity. Some of the other alleles investigated ( ce, ch, cp) as well as cch also had enzymes differing in heat stability. Heterozygotes of c/— with all other alleles displayed intermediate values of activities compared to homozygotes. These findings are important because they further support the view that the c-locus is the structural one for the enzyme.

22Studies by Winston and Lindzey ( 1964) and by Thiessen et al. ( 1970) have also demonstrated that albino mice differ from pigmented animals in a number of behavioral situations. Moreover, employing coisogenic subjects King and Rush ( 1976) have shown that albino mice consume significantly less alcohol and sleep (following injection of ethyl alcohol) significantly longer than either heterozygous ( C/c) or homozygous ( C/C) pigmented animals. These investigators also found that pigmented mice heterozygous for albinism ( C/c) tended to consume less alcohol and to sleep longer than those which were homozygous for the normal allele ( C/C). In this case, however, there was some disparity in the results suggesting a maternal effect and environmental influences.

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