Notes to Chapter 12

¹Deol ( 1970b) notes that there is a slight dilution of the coat in young heterozygotes, but that this disappears later on. Moreover, it should be noted that Wolfe and Coleman ( 1964) found that tyrosine incorporation was less in 5-day-old mi/+ than +/+ skin (see Figure 12-1).

²Although it is not possible to classify mi/+ and +/+ mice as easily later on Grüneberg ( 1948, 1952) states it can be done provided the eyes are examined with a strong light under magnification. However, according to him this classification "is complicated by the fact that contrary to statements in the literature, the color genes a, b and d also influence eye color very markedly." "In a normal ( A/A;B/B) mouse, the choroidal vessels are hidden from view by the choroidal pigment; the pupil is jet-black and the schlera next to the cornea is opaque and slate-grey." On the other hand, in cinnamons ( A/A;b/b) and in dilute ( d/d) animals the choroidal pigmentation near the fundus is somewhat diluted so that even adult animals show a dull red light reflex in their pupils. Grüneberg also states that a/a increases the amount of choroidal pigment; hence a chocolate mouse ( a/a;b/b)has much more pigment than a cinnamon ( A/A;b/b) animal. The influence which mi/+ has on the eye is similar to, but much stronger than, the diluting effect of b/b. "Thus a mi/+;A/A;B/B mouse shows a red pupillary reflex, and often the pigmentation is reduced in the periphery of the fundus so that the choroidal vessels are visible through a semi-transparent sclera near the corneal margin." In mi/+;A/A;b/b animals the pigmentation of the iris is also diluted so "that a ruby-colored reflex is generally visible in ordinary daylight with the naked eye." In Grüneberg's experience mi/+ can be distinguished from +/+ in the presence of A, a, B, b, D, and d on the basis of choroidal and iris pigmentation "except for rare errors in A/A;B/B or a/a;B/B mice" ( Grüneberg, 1952).

³Because the spotting effect of mi/+ was discovered accidentally, Grüneberg's analysis of this condition was, in part, a retrospective one. Consequently he was not absolutely certain whether any of the three animals he originally mated showed minor spotting. It should be emphasized, however, that the mi/+ male employed in this mating came from Hertwig's original strain, a strain in which tail spotting or spotting elsewhere does not seem to have occurred at all ( Grüneberg, 1948).

⁴Deol ( 1970b) found that all 20 ears he examined were affected. The abnormalities always were severe in the cochlea, but the saccule sometimes exhibited a fairly normal appearance, particularly in very young animals. In no instance was any section of the cochlea duct found to be normal, although the abnormalities were far from uniform throughout its length. The stria vascularis was abnormal in its entirety but small groups of hair cells occurred in the organ of Corti at scattered points. Degeneration of the spiral ganglion was not particularly severe, but this could have been attributed to the fact that no old animals were examined. Deol also observed the "severe dedifferentiation and cellular migrations occurred in the cochlear duct and the saccule in the majority of ears."

⁵Briefly "the microphthalmic mouse has a mild hypocalcaemia, hypophosphatemia, an increased serum alkaline phosphatase and greatly increased bone and serum citrate" ( H. Murphy, 1973). Although some animals are found with normal levels of these parameters, the severe osteopetrotic lesion is always present histologically. According to Murphy, 28% of the mice have a life expectancy in excess of 3 months which is much greater than the life expectancy of grey-lethal animals. As in the case of grey-lethal both thyroid and parathyroid endocrinopathies seem to be involved; at least many of the morphological, histological, and biochemical observations on these mutants can be explained by an increased secretion of calcitonin eliciting a compensatory increase in parathyroid hormone ( H. Murphy, 1973). The osteopetrotic condition in microphthalmic mice can also be improved by parabiosis with normal mice (Walker, 1972, 1973; Barnes et al., 1975) or by injecting cells from the spleen or marrow of normal animals into irradiated mutants (Walker 1975a, 1975c). Loutit and Sansom ( 1976) have been able to overcome the osteopetrotic defect of mi/mi mice by injecting them intraperitoneally at birth, or intravenously at weaning or maturity, with cell suspensions containing hematopoietic stem cells from normal syngeneic (or H-2 compatible allogeneic) donors. Thus following this treatment resolution of much of the osteopetrosis but none of the other effects of mi/mi occurred within a few months in the majority of cases. Moreover, irradiation of the recipients was not necessary to accomplish this "cure" ( Loutit, 1977). These investigators believe that "osteoclasis of scaffold-type woven bone is impaired in mi/mi" and "that osteoclastic cells are derived through circulating monocytes from hematopoietic stem cells." More recently Nisbet et al. ( 1978) have shown that if microphthalmic mice are either inoculated intravenously with bone marrow cells from allogeneic (but H-2 compatible) donors (bearing a T-6 marker), or are placed in temporary parabiosis with such animals, some improvement of their condition occurs (blindness and failure of eruption of teeth are unaffected) despite the fact that no donor cells can subsequently be found. On the basis of these findings they believe that during their limited period of residence the normal cells may cause considerable resorption of bone by providing osteoclastic precursors, or, they may trigger "the recipient's osteoclasts into effective function, comparable to the T-B cell-cell interactions of immunology." In contrast to the situation when normal cells are given to mi/mi mice, osteopetrosis can be produced by inoculating spleen cells from mutants into lethally irradiated normal recipients ( Walker, 1975b). Raisz and his colleagues ( 1977) have determined the response of mi/mi (and normal) long bones in organ culture to some known stimulators of osteoclastic bone resorption. Their results indicate that the congenital osteopetrotic condition stems from "a generalized defect in the function and hormonal response of osteoclasts and suggest that this cell line is separate from the osteoblast cell line which shows no impairment of hormonal response."

⁶The gene originally was given the symbol Wh but was changed to Mi^wh when its allelism with mi was demonstrated ( Grüneberg, 1953).

⁷When Mi^wh/+ is combined with splotch ( Sp/+) on a nonagouti background it produces a decided yellowing effect on the ventrum, almost like a^t, as well as some increase in white area. Although the combination Mi^wh/+;W^a/+ produces typically a black-eyed white phenotype on some genetic backgrounds some pigment occurs, primarily on the rump and around the ears (but demonstrably less than in Mi^wh/+;W/+) ( Hollander, 1959).

⁸Deol ( 1970b) found that pigment was present in about two-thirds of Mi^wh/+ ears but even in these cases it was severely reduced and mostly found in the utricle.

⁹The fact that Mi^wh/+ mice, which possess a good deal of pigment in the coat, display much severer inner ear abnormalities than in W^v/W^v animals, which are completely white, provides and excellent example of an exception to the rule that, in general, the abnormalities of the inner ear are related to coat color ( Deol, 1970b). On the other hand, as already noted (see Chapter 10, note 19), the correlation between abnormalities of the inner ear and pigmentation of the inner ear is quite good ( Deol, 1970b).

¹⁰Markert and Silvers ( 1956) did not observe any choroidal pigment in either a/a;Mi^wh/+ or a^t/a^t;Mi^wh/+ mice (see Table 10-1). However, only a few animals were examined. Indeed, Deol ( 1973) too found that in some Mi^wh/+ mice the entire choroid was unpigmented.

¹¹Deol ( 1971) found no melanocytes in the harderian gland of Mi^wh/+ mice. "although in sections a few granules could occasionally be seen which might have been lightly melanized melanosomes."

¹²The situation in the eye of Mi^wh/+ provides further evidence that the tissue environment can influence the amount of pigment produced (see Chapter 10, Section I, E). Thus while the pigment cells in the choroid of these genotypes form little or no pigment, their kindred cells in the outer layer of the iris in the same eye produce considerable amounts, enough to give it a normal appearance in most instances. As a consequence of these observations Deol ( 1973) concludes that there is evidently something in the tissue environment of the outer layer, presumably some melanogenesis-promoting factor or factors, which brings about a change in the behavior of the pigment cells contained therein.

¹³Nevertheless, according to Wolfe and Coleman ( 1964) there is some tyrosinase activity in 5-day-old Mi^wh/Mi^wh skin (see Figure 12-1) since the activity in this skin is greater than in albino ( c/c) and extreme dilution ( c^e/c^e) skin, skin in which tyrosinase is known to be absent or at very low concentration ( Coleman, 1962).

¹⁴Grüneberg ( 1953) points out that when the iris is pigmented, "careful inspection usually reveals the presence of a very slender pigment ring even through the closed eyelids at birth." He also draws attention to the fact that "the situation is like that in a normal 12-day embryo which has a pigment ring in the iris, but no melanin anywhere else."

¹⁵As pointed out by Deol ( 1970b) the fact that Mi^wh/Mi^wh mice are like W^v/W^v genotypes in terms of "external spotting" but much more like Mi^wh/+ as regards "internal spotting" again indicates that there is often little or no relationship between the two.

¹⁶Because Mi^wh/+ mice very occasionally display intensely pigmented "spots" (see Chapter 10, note 28), and because Hertwig ( 1942a, 1942b) noted that the tips of the whiskers of mi/mi homozygotes tend to be bent, one must consider the possibility that the mi-locus is a complex one involving, in addition to inviable melanoblast clones, two phenotypic colors of viable clones and two hair follicle phenoclones (see Mintz, 1971a). This conclusion is supported further by the interaction of certain mi-alleles, namely, Mi^wh with mi^bw, mi^sp, and mi^ws.

¹⁷Deol ( 1973) reports "in the iris the outer layer was unpigmented, but the inner layer always had some pigment, although it was unevenly distributed and much below normal in density." He also draws attention to the fact that the cells of this inner layer are derived from the retina which is almost completely unpigmented.

¹⁸Deol ( 1973) emphasizes the fact that because the identification of the amelanotic retinal pigment cell is beyond doubt, and very few of these cells in Mi^wh/mi heterozygotes form any pigment, that the widespread assumption that melanoblasts which fail to differentiate do not survive does not appear to be well founded, or at least is not invariably correct. While Deol's conclusion cannot be refuted this particular argument is not very convincing. Thus it is difficult to accept his premise that merely because the retinal cells of Mi^wh/mi fail to produce melanin they must be considered undifferentiated.

¹⁹Grüneberg points out that a similar situation is found in the dominant spotting ( W) series ( Chapter 10, Section I). The anemia of W/W animals is much more severe than that of W^v/W^v mice; yet W/+ heterozygotes are not anemic while W^v/+ mice are slightly so. Furthermore, W^v "dilutes" the fur when heterozygous with the normal allele and W does not. The basis for these different effects is not known.

²⁰As pointed out by Wolfe and Coleman (see also Hollander, 1968), the fact that mi^sp is expressed only when heterozygous with other alleles (but not +) makes it appear, at least superficially, like the t-T (tailless-Brachyury) relationship. The T/t complex is composed of a class of semidominant T mutations which, when heterozygous, produce a short tail and when homozygous are lethal. T mutations interact with recessive alleles at this locus to produce a tailless ( T/t) phenotype; this interaction makes it possible to detect recessive t alleles that would otherwise go unnoticed ( Artzt and Bennett, 1975; see D. Bennett, 1975).

²¹Wolfe and Coleman draw attention to the fact that mi was in the process of being backcrossed onto the C57BL/6J background when employed in these crosses, and that the faint dorsal pigmentation of mi/mi^sp animals present in early crosses was not observed in animals of this same genotype subsequent to generation 4, all hair being devoid of pigment at this stage.

²²Although it appears in Figure 12-1 that Mi^wh/+ may differ significantly from Mi^wh/mi^sp, this difference could be due to the fact that in some cases the pieces of Mi^wh/mi^sp dorsal skin assayed overlapped some faintly visible white spots which may have reduced the counts. Indeed, counts from Mi^wh/mi^sp were about the same as those from Mi^wh/+ in three samples in which white spotted areas were carefully avoided.

²³Since there are apparently no differences in the attributes of the pigment granules in mi^sp/+, mi^sp/mi^sp, and +/+ genotypes, one must account for the differences in the activity of tyrosinase in the skins of these animals. To account for this Wolfe and Coleman suggest that "there is a threshold level for tyrosinase incorporation, above which full pigmentation can occur." A similar suggestion was made by Coleman ( 1962) to account for the activity of the alleles at the albino ( c) locus. The data suggest that this threshold falls between 640 and 898 cpm (see Figure 12-1) compared to 600 cpm for the albino-locus series of alleles (see Table 3-5).

²⁴It should be stressed that although the pigment granules in Mi^wh/mi^sp hairs are yellow-brown when viewed by transmitted white light, they are nevertheless noticeably different from the small, round yellow granules produced by agouti locus alleles and by recessive yellow ( e/e).

²⁵Another example where mi-locus alleles when heterozygous produce an effect different than when either allele is homozygous occurs in Mi^wh/mi animals. Thus these heterozygotes are less microphthalmic than either parental homozygote ( Grüneberg, 1952).

²⁶An mi mutant also appeared spontaneously in the CBA/CaCrc inbred colony. Animals heterozygous for this mutation have less iris pigment than normal at birth. They also have nonpigmented tail tips and may display white spotting on the belly, but not the head. Homozygotes are devoid of pigment in the hair and eyes. The eyes are small and the eyelids do not open. The teeth of homozygotes fail to erupt and they are osteopetrotic. There is also a significant deviation from the expected segregation ratio when heterozygotes are mated, a deviation which appears to be due to the death of microphthalmic female embryos before or soon after implantation, Homozygotes of both sexes are sterile. In matings of heterozygotes with mi/+ mice, microphthalmic young occur which are indistinguishable from animals homozygous for the new mutation. It therefore seems likely that the new deviant represents a remutation to mi. Accordingly, it has been designated mi^Crc ( Hetherington, 1976).

²⁷In this regard it is of interest to note that in the tests with the Fk/+ males, both a slightly reduced implantation frequency and a somewhat higher rate of intrauterine death were observed. These, however, were not statistically significant.

²⁸One of the most significant influences on spotting occurred when A^y was substituted for a in the presence of W^j/+ as such a substitution reduced the amount of white in the coat from approximately 26 to about 4% ( Lamoreux and E. Russell, 1971). In the case of flexed-tailed, 95% of a/a;f/f mice displayed white spotting while only 23% of A^y/a;f/f animals had spots ( Lamoreux, 1973). Lamoreux also noted that neither lethal yellow nor recessive yellow had any influence on the amount of white spotting produced by patch ( Ph/+) and that a/a;p^un/p^un;bt/bt mice displayed less spotting than a/a;+/+;bt/bt animals.

²⁹Nevertheless, one mutation from c --> + did occur in a research stock not included in the analysis. This mutation is important because it provides strong evidence that albinism is not the consequence of a deletion.

³⁰Because the mutation of wild type to d at the dilute locus has a higher reversion than forward rate, Schlager and Dickie ( 1971) suggest that + --> d --> + changes are mediated by a base-pair change, whereas the + --> a or + --> c change may involve longer segments of the DNA chain.

³¹On the other hand, Lyon and Morris ( 1966) found no mutations in a different set of specific loci in over 9000 offspring. They suggest that the mutation rate of the specific loci usually used may be higher than average since the irradiation-induced rate was significantly lower in this new set of specific loci.

³²Fahrig ( 1975, 1977, 1978) has described an in vivo method, based on the pioneer experiments of L. Russell and Major ( 1957), for detecting genetic alterations in the somatic cells of mice. This test, known as "the mammalian spot test", involves treating mouse embryos, heterozygous for different recessive coat color genes, in utero, at 7-10 days gestation, with mutagens. These agents either are injected into the peritoneal cavity of the mother or are given to her orally. If this treatment results in an alteration, or loss, in a melanoblast of the wild-type allele of one of the genes under study a color spot is produced in the adult coat. Employing this test it has been found that (1) the frequencies of color spots in mutagen-treated animals depend upon the mutagens employed ( L. Russell and Major, 1957; L. Russell, 1977; Davidson and Dawson, 1976, 1977; Fahrig, 1975, 1977); (2) the position of a color spot seems to depend to a certain extent on its color; and (3) the more white-grey spots that are induced by a mutagen, the more spots are located on the ventrum. Fahrig ( 1978) also notes that the spontaneous frequency of color spots is very low and appears to be similar for different hybrids. For example, among 891 ( a/a;b/+;c^ch p/+ +;d/+;s/+) F₁ animals whose adult coat were examined, only 6 displayed spots (one a light brown head spot; one a white-grey spot on its underside; and four midventral white spots).