Notes to Chapter 10

¹An independent mutation to a W-allele occurred in the C3H/J colony in 1952. This mutation is in all respects indistinguishable from W and has been called W^x. In C3H/J, W^x/+ individuals have ventral spots of moderate size and no dorsal spots ( E. Russell et al., 1957).

²According to Grüneberg ( 1952), the number of m(W) genes involved in Dunn's experiments was probably fewer (about three).

³In a stock homozygous for the whole m(W) complex W is nearly completely dominant in its effects on the fur and W/+ x W/+ matings produce one-quarter W/W anemics (potentially white), one-half W/+ roan (nearly white), and one-quarter +/+ solidly colored animals. On the other hand, in a stock homozygous for the normal alleles of the m(W) complex, W is completely recessive and so heterozygous matings produce one-quarter W/W anemics (potentially white) and three-quarters solidly colored animals (two-thirds W/+ and one-third +/+).

⁴The black-eye in these animals is due entirely to the fact that the retinal layer of the eye is pigmented normally (see Chapter 1, note 5). It should be noted that the m(W) complex has no effect on W/W melanoblasts nor has it any apparent effect in W/W (or W/+) mice or erythropoiesis or gametogenesis.

⁵Thus the genotype W/+;s/s;p/p may be phenotypically indistinguishable from an albino ( c/c).

⁶The original W^v mutation occurred in a male of a strain of silvered black self mice who, unlike his sibs, had a number of small irregular white spots or streaks on his dorsal surface and a white streak on the mid-ventral line. The colored areas of his coat were also markedly paler than the typical coat color of the black silvered stock from which he originated. An independent mutation, indistinguishable from W^v, also occurred in the C57BR colony at the Jackson Lab in 1945 ( E. Russell et al., 1957).

⁷In 1952 Carter described a female mosaic mouse heterozygous for W^v which had two large patches of wild type hair, as if a somatic mutation had occurred from W^v to +. Nevertheless, when this animal was bred she produced a significant excess of W^v offspring indicating that a mutation in the opposite direction, i.e., from + to W^v, had occurred in her gonads. Carter discusses the possible explanations for this paradoxical situation and concludes that the most likely ones are somatic crossing-over (see Chapter 3, note 12) or somatic reduction at an early cleavage stage. If somatic crossing-over between W^v and the centromere had occurred both W^v/W^v and +/+ daughter cells would be formed; if the former cells had given rise to one ovary while the latter produced the wild type colored patches, this would explain the mosaicism. This seems a more likely explanation than somatic reduction at an early cleavage division, leading to haploid W^v and + cells, since it is most unlikely that a haploid ovary or haploid melanocytes could function successfully. Still another possibility, and one also discussed by Carter, is nondisjunction leading to aneuploid cells of genotype W^v/W^v/+ (going to the ovary) and + alone. However, the observed segregation of W^v and + in the offspring of this mosaic female does not agree as well with this explanation as with the other two.

⁸The harderian glands of these mice likewise possess melanocytes (see Table 10-1) ( Markert and Silvers, 1956).

⁹Another characteristic of W^v/W^v genotypes is that, unlike normal mice which are able to utilize both the D and L form of certain amino acids, they are unable to utilize the D form and this form is excreted. W^v/+ mice are more or less intermediate in this regard and excrete less of the D form (Goodman, 1955, 1956, 1958). The basis of this change in amino acid metabolism, and its significance, is not known.

¹⁰W^v/+ also was found to produce more white spotting than W/+ on four different genetic backgrounds. Furthermore, the amount of white spotting associated with each of these heterozygotes is greatly augmented when combined with Mi^wh/+ ( E. Russell et al., 1952).

¹¹Recently Heath ( 1978) has produced allophenics between mice carrying W^v and normal (+/+) animals of the AG/Cam strain. Six such allophenics have so far survived to adulthood and one of these is of the class W^v/W^v <--> +/+. The coat of this mouse displays the belly spotting characteristic of W^v/+ mice, whereas its blood is of the +/+ type and there is no evidence for transmission of mutant gametes. Two other mice are putatively W^v <--> +/+ allophenics. These mice too have belly spots but also show evidence of contributions from both partners in their blood and germ line.

¹²Schumann ( 1960) has presented evidence which she believes indicates that white spotting in mice is caused by a delay in the migration of melanoblasts from the neural crest. She contends that "as a result of this delay, the pigment cells do not reach some parts of the integument until after the skin has already reached such a stage of development that it is impervious to the melanoblasts" [see, however, Mayer and Maltby ( 1964) and Chapter 9, note 11].

¹³ W/W^v mice, like W/W and W^v/W^v animals, are severely anemic black-eyed whites. On most genetic backgrounds about one-half of these heterozygotes survive to adulthood ( E. Russell et al., 1957).

¹⁴One interesting result of this study was that whereas the combination of +/+ epidermis and W/W^v dermis always resulted in pigmented grafts, in only 50% of reciprocally combined grafts, i.e., W/W^v epidermis and +/+ dermis, did pigment occur. This variability in the occurrence of melanocytes in grafts in which the mesoderm is the +/+ melanoblast carrier undoubtedly reflects the migratory pattern of melanoblasts into the skin. As pointed out by Mayer ( 1973a), and in accord with other findings ( Mayer, 1973b), these results are interpreted best by assuming that melanoblasts initially migrate through the dermal mesoderm and secondarily, gain access to the ectoderm. Thus, by 13 days gestation the epidermis of +/+ embryos would be expected to be heavily populated with melanoblasts, which a few days previously occurred only in the dermis but which by now had, for the most part, left this layer (see Chapter 11, note 12).

¹⁵Gordon draws attention to the fact that the trace of pigment which occurred in one alleged W/+;+/+ <--> +/+;c/c allophenic was located at the tip of the right hind foot, an area which is consistently unpigmented in W/+ mice. He believes this suggests that the absence of pigment cells in this area of W/+ mice may not be due to a migratory insufficiency, but rather to the fact that W/+ pigment cells are marginally viable. He raises the possibility that "as cells migrate dorsolaterally in W/+ mice, cells more proximal to the dorsal midline may die, leaving empty spaces which are filled by backward migration of more distally located melanoblasts." In the W/+;+/+ <--> +/+;c/c mouse, however, these empty spaces may be filled by laterally migrating albino cells from a neighboring clone, thus permitting the W/+;+/+ cells to proceed to the tip of the foot.

¹⁶Mayer's observation that 61% of neural tube grafts (instead of the expected 25%) from W^v/+ matings did not produce any pigment when combined with neural crest-free +/+ skin may likewise be interpreted in terms of some inherent weakness of W^v/+ melanoblasts. Pn the other hand this observation also is in complete accord with Mintz's notion that some of the pieces of W^v/+ neural tubes, by chance, possessed only inviable melanoblast clones.

¹⁷Deol also found that the choroid of W^v/+ mice frequently to have "one or more moderately large unpigmented patches of an extremely irregular shape, the total unpigmented area being always less than half. The borders of these patches were sharp but heavily indented, somewhat like the skull sutures in old mice. The pigment, where present, seemed to be of normal density, an there was generally more of it in the ventral half than the dorsal. There was no tendency towards a regular pattern of symmetry."

¹⁸It is interesting to note that although the coat of W^v/W^v mice is invariably white, Deol ( 1970c) frequently found the inner ear of these animals to be pigmented. According to him in normal mice the distribution of pigment in the labyrinth is not uniform, but follows a characteristic pattern, occurring largely in the vestibular part. In the cochlea of +/+ mice pigment is confined to the stria vascularis, and the saccule is on the whole free of it. The free wall of the utricle of +/+ animals is heavily pigmented, as are certain well-defined areas in the semicircular ducts; pigment likewise occurs around the cristae, and among the nerve fibers, particularly near their external endings. In contrast, in the 33 W^v/W^v mice examined by Deol, 7 had no pigment in the inner ear on either side, 7 displayed it only on one side, and in 19 animals both sides were pigmented. Nevertheless, even in the 45 ears which possessed pigment its distribution was never normal. Thus, "the stria was always unpigmented except for a small region in the basal part in those few cases in which the basal stria was normal."

¹⁹Although Deol ( 1970b) notes that there is a good correlation between the severity of the effect which different spotting genes have on the inner ear with their effect on the pigmentation of this part of the ear, this does not always apply to animals of the same genotype. Thus, he found no clear correlation between the pigmentation of the inner ear and its abnormalities in W^v/W^v mice ( Deol, 1970c). Because of this Deol deems it possible that "the region of the neural crest which produces melanoblasts for the inner ear is different from that which produces the primordium of the acoustic ganglion."

²⁰The mean erythrocyte count in the normal young adult mouse is about 9-10 x 10⁶/mm³ whereas in the viable W^v/W^v it is about 5 - 5.5 x 106/mm³ ( E. Russell, 1954).

²¹ W/W^v mice also have been reported to have normal numbers of platelets and granulocytes in their peripheral blood ( J. Lewis et al., 1967) but decreased numbers of megakaryocytes ( Chervenick and Boggs, 1969; Ebbe et al., 1973a; Ebbe and Phalen, 1978) and neutrophils ( Chervenick and Boggs, 1969) in their marrow. Moreover, although these mice respond very poorly to erythropoietin ( Keighley et al., 1966), they respond as well as +/+ animals to lowered oxygen tension by developing elevated hematocrits, reticulocytosis, and increased blood volume ( Keighley et al., 1962; Bernstein et al., 1968). In fact during prolonged periods of hypoxia such mice produce large amounts of erythropoietin but, because of their genetic defect in RBC precursors, they remain anemic ( E. Russell and Keighley, 1972; see also Fried et al., 1967). Erythropoiesis is also stimulated in W/W^v mice by bleeding ( Grüneberg, 1939; Harrison and E. Russell, 1972).

²²Recently Flaherty and her associates ( 1977) have demonstrated that the cell surface antigens of normal erythrocytes change during their maturation and that the appearance and disappearance of these antigens differ in +/+ and W/W cells. Employing both a rat anti-mouse erythroblast serum and a rat anti-mouse adult RBC serum they found that the former antiserum recognized antigen(s) present on erythroid cells early in development, while the latter recognized antigen(s) present on mature erythroid cells only. Relative to normal cells, the erythroid cells of W/W mice were out of phase; the developing cells prematurely lost the "early" antigen(s) recognized by the antierythroblast serum and prematurely gained the "late" antigen(s) recognized by the anti-RBC serum. Flaherty and her colleagues suggest "that the W-locus may affect the erythroid cell surface in such a way as to preclude normal recognition of the mitogenic signals needed for continued proliferation." Such a cell surface defect could, of course, be either a primary one or one which results from some abnormal differentiative process in these cells which then leads to an altered makeup of the cell surface. For other hematological investigations on W/W^v mice see Till et al. ( 1967), Boggs et al. ( 1973) and Harrison ( 1972b, 1975a, 1975b).

²³In addition to the original W^v/W^v deviants of Little and Cloudman which occasionally were fertile, a stock displaying fertility of some homozygous W^v mice was produced by transferring this allele into a stock of W/+ animals selected for prolonged survival of W/W anemics ( E. Russell and Lawson, 1959). Following this procedure the incidence of fertile W^v/W^v males was much higher than the incidence of fertile W^v/W^v females. Indeed only 2 of 14 were fertile and they produced only one small litter each ( Mintz, 1960). This sex difference is not unexpected since, in contrast to the situation in males, any increase in the number of germ cells in the female can occur only before meiotic prophase sets in. Thus, as emphasized by Mintz ( 1960), if conditions favoring germinal proliferation are slow in operating, the female is at a marked disadvantage as compared with the male. It should also be noted that in fertile W^v/W^v adult males the testis does not present a normal histological picture. Although normal-looking regions are evident, spermatogenic elements may be lacking in some cross-sections of tubules and even where spermatogenesis has clearly been occurring, it may be arrested or abnormal ( Mintz, 1960).

²⁴E. Russell and Fekete ( 1958) also observed that these W^v/W^v females displayed diminishing numbers of young and atretic follicles until 5 months of age. This diminution was accompanied by an invagination of the germinal epithelium, culminating by the seventh month in the formation of tubular adenomas of all ovaries. Similar findings have been reported for the ovaries of other W-mutant heterozygotes ( W^x/W^v and W^j/W^v) which progressed through stages of tumorigenesis classified as tubular adenomas, complex tubular ademonas, and finally granulosa-cell tumors and luteomas ( E. Murphy and E. Russell, 1963). It is thought that tumors develop in these females as a consequence of the lack of developing ovarian follicles, i.e., due to the deficiency of germ cells there is an underproduction of estrogen and an overproduction of pituitary gonadotropin with excess stimulation of the gonad. That nothing is wrong with the pituitary of W^v/W^v mice is demonstrated by the fact that when ovaries from normal (+/+) C57BL females are implanted into the ovarian capsules of weaning-age W^v/W^v females, they function normally. Such recipients mate and rear healthy normal litters. This indicates that the W^v/W^v pituitary can react normally to the estrogens from the +/+ gonad, and that there is also a normal response of the W^v/W^v uterus and mammary gland to this pituitary stimulation ( W. Russell and E. Russell, 1948).

²⁵According to Mintz ( 1957a), primordial germ cells are first observed in the normal mouse embryo at 8 days "when they are seen in the yolk sac splanchnopleure, caudal end of the primitive streak, and root of the allantois. Migration through neighboring tissues begins at 9 days, when the cells occur in the gut splanchnopluere and may proceed up the dorsal mesentery to the gut. At 10 days they are found in the mesentery and at its root, near the dorsal aorta, around the coelomic angles, in the mesonephric regions, and in the paired germinal ridges." Migration is largely completed at 12 days (see also Chiquoine, 1954; Mintz and E. Russell, 1957).

²⁶Inasmuch as the experimental matings in this study were between heterozygotes, they should theoretically yield 25% defective offspring. The actual frequency of embryos with a severe paucity of germ cells was 28-29%. It should also be noted that there were no apparent differences in the number of germ cells in W/W, W/W^v, and W^v/W^v embryos. This is interesting because, as shown by Coulombre and E. Russell ( 1954), at 0-28 days postpartum the gonads of surviving "defective genotypes" can be ranked with W/W gonads exhibiting the most severe defect and W^v/W^v the least. Thus it appears that the different effects which W and W^v have on germ cell development occurs between the end of germ cell migration and parturition ( Mintz, 1957a).

²⁷ W/W^v mice are also very deficient of mast cells; their skin possesses less than 1% the normal number, and they do not occur in other tissues ( Kitamura et al., 1978). After transplantation of bone marrow cells from normal (+/+) donors, however, the number of mast cells in the skin, stomach, caecum, and mesentery of these mice increases to normal levels ( Kitamura et al., 1978). Although the fact that W/W^v genotypes lack both melanocytes and mast cells seems to be consistent with Okun's ( 1976) claim that these cells have a common precursor, as pointed out by Kitamura and his associates, this contention is inconsistent with the observation that the white spots of W^v/+ and W/+ mice have as many mast cells as their pigmented dorsal skin. W/W^v mice also appear to be deficient of a thymus derived cell. Thus when they are injected with normal (+/+) bone marrow cells that have been treated with antiserum to the thymus cell antigen theta (Thy-1) and complement (C'), their anemia is not cured. The addition of +/+ thymocytes to these cells, however, restores their capacity to cure the anemia ( Wiktor-Jedrzejczak et al., 1977). These findings suggest that a theta-sensitive cell is required for the promotion of mouse hematopoietic stem cells into erythrocytes, and that W/W^v mice are deficient of such a cell ( Wiktor-Jedrzejczak et al., 1977)

²⁸Schaible ( 1969) reported that Mi^wh/+ and Va/+ heterozygotes also display a similar type of variegation with no evidence of germinal mosaicism and that "reversion to the non-mutant color occurred in 5.3, 8.0 and 90.5%, respectively, of 4083 Mi^wh/+, 1738 W^a/+ and 317 Va/+ individuals from the stocks having minimum white."

²⁹In 1953 Strong and Hollander published a paper entitled "Two Non-Allelic Mutants Resembling " W" in the House Mouse." One of these mutants, designated as "type-1," originated in the N-strain which is dilute, brown, nonagouti, and piebald ( d/d;b/b;a/a;s/s). This mutant displayed more dilute pigmentation and extensive white areas, and breeding tests indicated it "was of the W-type, the homozygote being anemic and dying young." In general, however, the heterozygotes were more like W^v in phenotype. The second mutant designated as "type-2," appeared in a strain called 3CAMG. This mutant "showed a large frontal blaze, a large patch on the belly, and several spots on the shoulders and back." When these "type-2" animals were mated to each other it became evident that homozygotes for the mutation did not survive until birth. A subsequent examination of fetuses from these matings revealed that at 15 days some were anemic and some were already dying. Crosses between "type 1" and "type2" indicated that they were not alleles, and crosses between "type-2" and W/+ mice produced no anemic young but the phenotypes of the F₁ and the 2:1 ration obtained indicated intrauterine lethality. Unfortunately "type-1" was never crossed with a known W-allele. Nevertheless, "type-2" evidently was the consequence of a W-locus mutation and accordingly has been designated W^s for Strong's dominant spotting ( Ballantyne et al., 1961).

³⁰A spontaneous mutation in the CBA/H inbred strain has been reported ( Searle et al., 1974) which could very well be a remutation to W^b. On an agouti background, animals heterozygous for this mutant have a large white frontal blaze, much white spotting on the body, a general lightening of the coat, and a white belly. They are easily distinguishable from W^v/+ littermates. Compounds with W^v are black-eyed whites which survive to maturity. Animals homozygous for this mutation are very anemic black-eyed whites which do not survive for more than about 10 days.

³¹In addition, a separate analysis revealed that the incidence and the size of the white spots on the crown of the head of W^j/+ mice was significantly greater than in W/+ animals, when both heterozygous genotypes were on the same genetic background ( E. Russell et al., 1957).

³²This "triplet" is likely a "quartet" as more recently Southard and M.C. Green ( 1971) have reported that another white spotting determinant, recessive spotting ( rs), also appears to be very closely linked to W^v. rs was originally described by Dickie ( 1966b). It was first noticed when two animals with large head blazes and large belly spots and diluted bellies were found in a C3H/HeJ litter. Although breeding tests indicate that rs is a viable recessive mutation, nevertheless, 9-21% or rs/+ animals have a long thin white spot on the ventrum (but no head blaze and no dilution). Both W^v +/+ rs and sl/+;rs/+ genotypes are black-eyed whites, fertile, and nonanemic.

³³At 9 days of gestation the Ph/Ph embryos can be distinguished from their normal sibs by external inspection. They all display wavy neural tubes. The more severely affected embryos have irregularities of the somites and the most abnormal individuals are inflated with enormous hearts, or hearts of about normal size inside a very large pericardium. Depending on the degree of abnormality, Ph homozygotes are retarded to a greater or lesser degree as compared with their normal littermates ( Grüneberg and Truslove, 1960)

³⁴Nevertheless there is some evidence that when the amount of white spotting is increased in Ph/+ mice by selection, there is a concomitant decrease in fertility ( Truslove, 1977).

³⁵In contrast to Ph, where the correlation in the amount of white spotting between parents and offspring is incomplete ( Grüneberg and Truslove, 1960), in Ph^e mice it appears to be complete ( Truslove, 1977).

³⁶When the tail is partly pigmented the tip frequently has black hairs as well as pigmented skin while the proximal portion may have pigmented skin with unpigmented hairs.

³⁷Also, the scrotal skin of Rw/+ males remains dark in spite of the fact that the hair arising through it as well as the surrounding skin and hair are unpigmented. In females the perianal skin likewise is pigmented. In contrast, in Ph/+ males when the ventral white area includes a portion of the scrotal region the scrotum itself lacks pigment. Moreover, unlike Ph/+ the size of the interfrontal bone of Rw/+ mice is normal.

³⁸ Ph/+ mice may also have fewer viable melanoblast clones than Rw/+ animals and this too could contribute to the deficiency of epidermal melanocytes in the unpigmented (and pigmented) regions of their skin.