Notes to Chapter 2

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1Throughout this book the wild-type allele is designated sometimes by + or, for the sake of clarity, sometimes by its alphabetical symbol. Thus + = B, C, D, P, S, w etc.

2Cizadlo and Granholm ( 1978a) have histologically examined embryos derived from Ay/a x Ay/a and a/a x Ay/a matings at 105 hours postcoitum. Their observations confirm the suggestion of Calarco and Pederson ( 1976) that the cells of the inner cell mass are more susceptible to the effects of Ay/Ay than are trophoblast cells, although both populations eventually succumb. Their findings are also in accord with Eaton and M.M. Green's ( 1962, 1963) conclusion that failure of trophoblast giant cell proliferation in Ay/Ay embryos is the cause of the lethality, and with Pederson ( 1974) and Calarco and Pederson's ( 1976) conclusion that the effects of Ay homozygosity occur over an extended period of time. Thus individual blastomeres may become arrested as early as the 4- to 8-cell stage with "normal" development proceeding in the remaining cells until the late blastocyst stage. Finally, their observations are also consistent with Pedersen and Spindle's ( 1976) report that presumed homozygous Ay blastocysts do not develop as fast and possess fewer cells than normal littermates when cultured from cleavage stages. (See also Cizadlo and Granholm ( 1978b) and Granholm and P. Johnson ( 1978).)

3Heston and Vlahakis ( 1961b) have shown that the effect which Ay has in increasing the incidence of methylcholanthrene-induced pulmonary tumors seems to be related to its effect of the growth rate. Thus they found that by restricting the food intake of yellow mice they were able to reduce their susceptibility to these tumors to the level of nonyellow ( a/a)—but otherwise genetically identical—mice. From experiments based on the graft-versus-host reactivity of spleen cells, Gasser and Fischgrund ( 1973) observed that the immune responsiveness of Ay/a (and Avy/a) mice may be impaired and suggest that this may be involved in their higher tumor susceptibility.

4Some of the pigment patterns displayed by the hairs of "agouti" Ay/a phenotypes are strikingly different from the standard agouti pattern. These aberrant pigment patterns are especially apparent among the awls ( Galbraith and G. Wolff, 1974).

5This association of the yellow phenotype with obesity in both Aiy and Avy genotypes led Dickie ( 1969a to suggest that those environmental factors which stimulate phaeomelanin synthesis may block or inhibit a pathway used in normal fat metabolism (but see Section II, A, 2).

6Although A is usually regarded as the wild-type allele of this series by many authors, according to Grüneberg ( 1952) this is an oversimplification. Thus he notes that the Western European house mouse (M. m. domesticus Rutty) usually is grey-bellied, but sporadic cases of Aw are by no means rare, and there are entire populations in which this allele has replaced A ( Clarke, 1914). Moreover, according to Schwarz and Schwarz ( 1943) all wild subspecies of M. musculus are Aw, and the dark belly of A is the hallmark of commensalism. It is therefore evident that one can equally justify Aw and A as the wild-type allele of the series.

7Agouti (and white-bellied-agouti) mice also possess some all-black (or in b/b animals, brown) hairs on the dorsum. Thus, according to Galbraith ( 1969), of the four main hair types which comprise the dorsal pelage, zigzags are invariably banded, auchenes are usually banded, and monotrichs are never banded. Awls, on the other hand, may be either banded or totally black.

8"Pseudoagouti" am/a mice are indistinguishable from "pseudoagouti" Avy/a animals except by breeding tests.

9Very recently G. Wolff ( 1978) has published the results of extensive breeding experiments which indicate that the phenotype expressed by viable yellow ( Avy) and mottled agouti ( am) genotypes is significantly influenced by the agouti locus genotype and the genetic background of the dam. Thus in a reciprocal cross between a C57BL/6 ( a/a) subline and the strain AM/Wf, which is homozygous for am, he found that 29.5% of the offspring of the C57BL/6 mothers were of the "pseudoagouti" phenotype whereas no animals displaying this phenotype were produced by the AM dams. Wolff also found in two different reciprocal crosses using four different inbred strains, that the proportion of Avy/a offspring of "pseudoagouti" phenotype differed according to the strain of the mother. He also found in Avy/a x a/a reciprocal matings that, regardless of strain, mottled yellow Avy/a females produced significantly fewer "pseudoagouti" Avy/a young than did black ( a/a) females. Although Wolff does not consider the possibility that his results may reflect some maternal influence on the selection of phenoclones, in the light of Mintz's observations (see Chapter 7), this seems quite possible.

10Although As was suspected of being an inversion because it suppressed crossing-over between itself and pallid ( pa) ( R.J.S. Phillips, 1970b) while increasing crossing-over between a and brachypodism ( bp) ( R.J.S. Phillips, 1968), it was not until recently that this was confirmed. Thus E.P. Evans using G-banding has demonstrated the presence of an inversion of chromosome 2, involving about 40% of the chromosome, extending from about the T7Ca breakpoint proximally to the A-locus distally ( Evans and R.J.S. Phillips, 1978). This raises the possibility that other "alleles" of the agouti series may also represent structural alterations. In this regard R.J.S. Phillips (personal communication) suggests that nonaouti lethal ( al) could be a deletion, and agouti umbrous ( au), which so far has shown no crossing over with bp in 935 animals, may also be structural. ax too belongs in this category.

11A phenotypically similar umbrous factor as As, known as a6H, has been described by Searle ( 1966, 1968a; see also Batchelor et al., 1966). This factor arose in a neutron irradiation experiment and seems to be inseparable from a reciprocal translocation [known as T(2;8)26H] and so may exemplify a position effect. a6H/a animals are dark umbrous agouti with light pinna hairs. a6H/A are normal agouti, and a6H homozygotes are dark agouti with dark pinna hairs.

12As emphasized by Wallace ( 1965) this theory of pseudoallelism does not preclude the idea that different regions of the body may vary in their capacity to respond to some alleles. It also remains to be determined how many "mini-loci" are involved and the exact parts of the pelage they control. Epistasis between some pseudoalleles must be considered too.

13Wallace ( 1965) also points out that whereas the genotypes au/au and ae/ae have dark ears, the alleles at and a, which stand between them in the series, produce lighter ears, both homozygously and in compounds with au and ae. This, she believes, can best be explained by assuming that there is a locus (say, E) which controls ear color, with dominance of the yellow component. Thus au and ae are e, and at and a (and all the other alleles) are E.

14Dickie ( 1969a) found that the most common spontaneous mutational events at the agouti locus involve heritable and nonheritable changes from a to at and from a to Aw. Thus at least 31 heritable mutations from a to at (20 in the C57BL/6J strain) and at least 17 heritable mutations from a to Aw (6 in C57BL/6J) occurred at the Jackson Laboratory between July 1, 1961 and January 1, 1968 (see Grüneberg, 1966a)

15This situation occurred in a C57BL/6J male. The deviant was phenotypically light-bellied agouti but, when mated to a variety of unrelated nonagouti females, produced three classes of offspring. Some were light-bellied agouti, some were black-and-tan, and some were nonagouti in a ratio of 1:1:2. Extensive testing of all light-bellied agouti, black-and-tan, and some nonagouti offspring indicated that Aw and at behaved similarly to other known reoccurrences of these mutations. No animals of the genotype Aw/at were detected nor did cytological studies reveal any chromosome abnormalities. Inasmuch as the three classes of phenotypes were produced in almost a perfect 1:1:2 ratio, it seems most likely that two mutations had occurred at a very early stage of development ( Dickie, 1969a).

16To overcome the difficulties inherent in both the "complex" and "single-locus" theories, R.J.S. Phillips ( 1966a) has discussed possible models in which the agouti locus would consist of several genes in an operon-like organization. On the one hand, each cistron could relate to one part of the body and all be controlled by an operator. On the other hand, the structural genes could individually be controlled by regulatory genes. In one model she proposes that As, which appears to act coordinately on the other alleles of the locus, could be assumed to be an allele of the operator or to be in the first cistron, and to affect transcription through the rest of the complex. According to this scheme ae might act similarly whereas atd could be an allele in a subsequent cistron and influence only the transcription of a "banding" region. In another model she suggests that As and ax, alleles which are known to show crossing over with the rest, could be considered as mutations of the operator controlling the cistron, whereas the other alleles would be structural mutants affecting the amount of some substances on which the synthesis of phaeomelanin depends.

17It should also be noted in Table 3-1 that this reduction in tyrosinase incorporation by Ay was of the same order regardless of the genetic constitution at either the b-locus or c-locus (i.e., cch/cch) ( Coleman, 1962).

18Prota ( 1972; see also Prota and Nicolaus, 1967; Fattorusso et al., 1969; Misuraca et al., 1969; Flesch, 1970; Prota and R. Thompson, 1976) has provided evidence from his avian studies that phaeomelanins are amphoteric pigments produced in vivo by a deviation of the eumelanin pathway involving a reaction between cysteine and dopaquinone, produced by the enzymatic oxidation of tyrosine. He has also shown that this reaction involves 1,6-addition of cysteine to dopaquinone to form two new amino acids, 5-S-cysteinyldopa and 2-S-cystinyldopa, and that under physiological conditions these intermediates are converted rapidly into dihydrobenzothiazine derivatives whose further oxidation gives rise to phaeomelanins (see Figure 2-3). Multivesicular bodies have been implicated in the formation of both eumelanosomes and phaeomelanosomes. Thus investigations support the view that the fusion of a Golgi-derived tyrosinase-containing vesicle with a vesicle from the endoplasmic reticulum is involved in the genesis of these granules (see Bagnara et al., 1979). Prota ( 1970) also contends "that the so-called tricosiderins, the red pigments of human hair, are chemically and biogenetically related to phaeomelanins." It should also be noted that whereas three bands of tyrosinase activity are detected in acrylamide gel electrograms of extracts of eumelanin pigmented skin (see Chapter 3, Section I,I), extracts of Ay/— skin give rise to only a single band, the intensity of which is attenuated when compared to that of other genotypes ( Holstein et al., 1967, 1971; see also Geschwind and Huseby, 1972; Geschwind et al., 1972). This indicates that there are "marked quantitative and perhaps qualitative differences between tyrosinases associated with eumelanogenic and phaeomelanogenic melanocytes" ( Holstein et al., 1971). Indeed, Holstein and his colleagues contend that this observation does not necessarily support a switch mechanism which is based only on the prevailing titer of cysteine. They believe the observed differences in tyrosinase could "be related to functional properties of the enzyme involved in shunting the melanogenic mechanism either toward the production of eumelanin or of phaeomelanin."

19Takeuchi ( 1970) has also investigated the activity of the agouti locus in vitro. He found that when a piece of dorsal A/A skin from a 2-day-old mouse was cultured, no phaeomelanin was produced. On the other hand, the formation of yellow pigment was observed when skin from a 3-day-old agouti animal was maintained in culture for 4 days. Addition of dopa to the culture medium of 2-day-old A/A skin resulted in the formation of phaeomelanin. However, phaeomelanin synthesis was prevented in 3-day-old A/A explants maintained with excess tyrosine. On the basis of these observations Takeuchi suggests that the formation of the agouti pattern may depend upon a regulatory system of the feedback type, involving both the a- and c-loci. His hypothesis is discussed in some detail by Holstein et al. ( 1971).

20According to Geschwind and colleagues ( 1972) the few melanocytes which occur in the dermis of the tail and scrotum as well as in the connective tissue of the nipple of Ay/— mice contain yellow granules, i.e., phaeomelanin. This observation, however, remains to be confirmed.

21This influence of tract specificity not only holds for black-and-tan and yellow-bellied agouti genotypes but for nonagouti animals as well. For example, the hairs round the mammae of a/a mice contain yellow pigment and if this region is included in a/a;ce/ce grafts, all potentially intensely pigmented ( C/—) melanocytes which invade these follicles produce some phaeomelanin, i.e., they produce the characteristic type of pigmented hair for this region.

22Not only do mouse melanoblasts respond to the agouti locus genotype of mouse hair follicles but rat melanoblasts can likewise respond to the agouti locus genotype of these follicles. This was demonstrated by transplanting agouti (but albino) BALB/c ( A/A;c/c) mouse skin from 18- to 20-day-old fetuses to immunologically tolerant newborn (Lewis x BN)F1 ( a/a;C/c) rats. About 2 weeks following this procedure some pigmented hairs, displaying the typical agouti pattern, were observed within the borders of the mouse xenografts (Silvers, 1965).

23One wonders whether this is responsible for the fact that the number of melanocytes in the ear skin of Ay/a mice appears drastically reduced from the number in a/a, A/— or at/— ears. Moreover, a similar difference has been reported in Ay/A vs A/A plantar skin ( Quevedo and J. Smith, 1963) and dorsal body skin ( Quevedo and McTague, 1963) after exposure to ultraviolet light (UV). Whereas the body skin of lethal yellow ( Ay/A;B/B) mice tans poorly in response to UV, the skins of black ( a/a;B/B) and black agouti ( A/A;B/B) genotypes tan noticeably. Following UV exposure only a few dopa-positive melanocytes occur at the dermoepidermal junction and only a few melanin granules are released to malpighian cells in Ay/A;B/B mice. On the other hand, in the skin of A/A;B/B animals there is a marked increase in the number of dopa-reactive epidermal melanocytes and in the number of melanin granules within malpighian cells ( Quevedo et al., 1967). The tanning response of viable yellow ( Avy/a;B/B) mice varies with the phenotype. In general, the skin of "clear yellow" Avy/a mice and the "clear yellow" areas of mosaically pigmented animals present a histological picture similar to that found in similarly exposed lethal yellow ( Ay/A;B/B) mice, while the skin of "agouti" Avy/a animals and the "agouti"-pigmented areas of mosaics respond like A/A;B/B genotypes ( Quevedo et al., 1967).

24According to Galbraith ( 1964) at about 6.5 days after plucking the dorsal hairs of adult agouti mice, many follicles begin to transition from synthesis of eumelanin to production of phaeomelanin and at about 9 days postplucking the reverse process is most evident. This switch from production of black pigment to yellow pigment is exceedingly rapid.

25Galbraith and Arceci ( 1974) found that individual zigzag hair bulbs contained from 5 to 18 melanocytes and that the average number of these cells in each hair bulb ranged from 10 at day 7 in both black and yellow mice, to 12 in yellow and 13 in black animals at day 13. As noted by these investigators these values are a little higher than Chase's ( 1951) estimate of 4 to 8 melanocytes per zigzag follicle and are lower than, but within Potten's ( 1968) observed range of a mean 7 to 22 melanocytes per bulb in the Strong F ( a/a;b/b;cch/cch;d/d;s/s) mouse strain.

26This mutation was designated as So until its allelism with e was established by Searle ( 1968b).

27It appears that the phenotypes of the A/A;Eso/E mice described by N. Bateman and the A/ae;Eso/E animals of G. Wolff et al. were slightly different. Whereas the former investigator noted that the flanks of his heterozygotes were "flecked with yellow hairs," the latter investigators found no evidence of phaeomelanin in their heterozygotes. It is also not clear whether the "yellowed hairs" which Bateman observed on the perineum of Eso homozygotes and heterozygotes resulted from the occurrence of phaeomelanin.

28Poole and Silvers (unpublished) have produced lethal yellow sombre ( Ay/a;Eso/E) mice and they too are completely black with no yellow pigment and show the same tendency toward obesity as "yellow lethal yellows."

29Wolff et al. also observed that zigzags of A/ae;Eso/E mice seemed to be more intensely and uniformly pigmented than those of Avy/A;Eso/E animals.

30As pointed out by Wolff et al. a similar black and white "banding" pattern occurs, albeit infrequently, among hairs of phenotypically agouti (A/A) animals ( Galbraith, 1964).

31For details concerning the distribution, morphology, biometrical characteristics, and cytogenetics of this subspecies see Gropp et al. ( 1969, 1970).

32Accordingly its designation was changed from Tob to Etob.

33According to Searle and Beechey ( 1970), because young e/e mice are very sooty it is difficult, but not impossible, to discriminate between young e/e and +/+ mice on dilute ( d/d) or leaden ( ln/ln) backgrounds. They also note that the degree of dilution of yellow areas in Ay/—;d/d and e/e;d/d mice of similar sootiness is about the same; that chinchilla ( cch) removes less yellow from e/e than from Ay/—, so that it is hard to differentiate between e/e;cch/cch and e/e;C/— mice before weaning age; and that the ears of Ay/— mice usually look a little lighter than e/e ears, presumably because of the frequently greater sootiness of the latter.

34Although this comparison was made employing hairs from a/a;B/B;e/e and Ay/a;b/b;E/E genotypes, it does not seem likely that the difference at the b-locus was responsible for the disparity since E. Russell ( 1948) obtained nearly identical counts for cortical phaeomelanin granules in Ay/a;B/B and Ay/a;b/b hairs.

35There is some evidence that total tyrosinase activity is greater in e/e than in Ay/a skin. This evidence stems from the observation of Geschwind et al. ( 1972) that tyrosinase specific activities in 90-day-old e/e skin was about double that of Ay/a skin.

36Nevertheless, Pomerantz and Chuang ( 1970) found that injections of beta-MSH not only produced a 45-50% increase in tyrosinase levels when administered to neonatal black or brown mice but the former, but not the latter, animals became darker than untreated controls. Hirobe and Takeuchi ( 1977a, 1977b) too have shown that if neonatal C57BL/10J ( a/a) mice are inoculated subcutaneously with alpha-MSH or with dibutyryl cyclic AMP, or if the skin of these mice is exposed in vitro to these agents, the number of dopa-positive melanocytes in the epidermis increases (see also Hirobe and Takeuchi, 1978). They believe this increased activity requires de novo transcription and translation since it can be suppressed by actinomycin-D or cycloheximide.

37In some of these studies Ay/— animals from 75-90 days of age were plucked (to stimulate hair growth) 8 days prior to receiving a subcutaneous injection of MSH [0.05 ml of a solution of natural or synthetic alpha-MSH (3 mg/ml) suspended in beeswax peanut oil]. Twelve to 24 hours following this inoculation skin biopsies indicated a gradual shift from phaeomelanin to eumelanin production in follicular melanocytes. This production of black pigment seemed to reach its peak approximately 36 hours after the injection and the black granules predominated until some time between the 72nd and 96th hour postinoculation when phaeomelanin synthesis resumed. Geschwind and his associates also demonstrated that the kinetics of the appearance of eumelanin in the hair follicles of these treated mice, as determined by histological sections, was generally consistent with the increase in tyrosinase activity. Thus a 2.5- to 5-fold increase in tyrosinase activity was produced in plucked Ay/— skin within 24 hours of MSH administration, and similar large increases in tyrosinase activity were produced within 24 hours of injecting the hormone into 4-day-old Ay/— mice. In Ay/— mice treated with hormone for a prolonged period an increase in dermal melanocytes as well as a significant increase in the number of melanosomes in the epidermis of the nipple, tail and scrotum was also observed ( Geschwind and Huseby, 1972). Finally it should be noted that while in preliminary experiments Geschwind and Huseby found that both cycloheximide and colchicine prevented darkening of MSH-treated lethal yellow mice, puromycin and actinomycin-D did not (but see note 36).

38Since sombre ( Eso) produces intensely black phenotypes, even in the presence of Ay/—, Geschwind and his associates ( 1972) determined whether Eso normally exerted its effect by increasing MSH levels in sombre mice. As might have been expected from the observation that MSH had no effect on altering the color of e/e mice, assays of plasma from A/—;Eso/— mice revealed no elevation of melanocyte stimulating activity levels when compared to levels in A;E/E animals.

39Because such very dark sables appear to have a glossy black back and a yellow belly, they came to be known as "black-and-tans." This designation should not be confused with the genuine black-and-tan caused by the agouti locus allele, at.

40This umbrous effect can be recognized also in Ay/—;b/b and A/—;b/b mice ( Grüneberg, 1952).

41When some wide crosses between strains are made and selection is practiced toward either darkening or making lighter the A/— segregants, the results described by Dunn can still be obtained and indicate a considerable potential reservoir of modifiers, i.e., polygenes (Chase, unpublished).

42The recombination between mg and a was 9.8 +/- 1.2% in males and 13.2 +/- 1.0% in females. This sex difference is significant and is in the same direction as that observed by Fisher and Landauer ( 1953) for this region of the second chromosome ( Lane and M.C. Green, 1960).

43 md, by virtue of its association with Robertsonian (whole arm) translocation (16.17)7Bnr (hereafter Rb7), has been shown to be located close to the centromere on chromosome 16 ( Roderick et al., 1976).

44Because md was shown to be "0%" from the centromere of chromosome 16, R.J.S. Phillips and G. Fisher (personal communication) crossed mice homozygous for nc to animals carrying Rb7 and so far have found 0/50 recombinants between nc and Rb7 in the appropriate backcross. From this observation it appears that nc is closely linked to the centromere of either chromosome 16 or 17 and, on the basis of other information, it has been located on 16.

45On the other hand, recessive yellow nonagouti curly ( e/e;nc/nc) mice are yellow with curly whiskers indicating that e is epistatic over nc with respect to coat color ( Beechey and Searle, 1978).

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