Chapter 10

Dominant Spotting, Patch, and Rump-White

I. Dominant Spotting ( W-Locus)

There are probably no coat color determinants which have received more attention than the alleles at the W-locus (chromosome 5). Most of this attention, however, has been devoted to the influence which two alleles at this locus, W and W^v, have on hematopoiesis for, like the steel ( Sl) series of alleles (see Chapter 11, Section I), the W-series too affect erythropoiesis and gametogenesis. While it is beyond the scope of this chapter to review in detail all the investigations which have been carried out on the nonpigmentary manifestations of these genes, they must nevertheless be given some consideration as they could be related to the influence of the locus has on coat color. Consequently, after considering W and W^v's influence on pigment formation, a more general account of their other consequences will be reviewed before the effects of the other alleles at the locus are described.

A. The Influence of W on Spotting

The influence of W on white spotting has been well reviewed by Grüneberg ( 1952) and much of this section is based upon his treatment of this subject. W was originally recognized by Durham ( 1908) and initially studied by Little ( 1915) and by Sô and Imai ( 1920) who demonstrated that it was lethal when homozygous.

W/+ mice generally have a well-defined belly spot with sharp edges (see Figure 11-1) and a very variable amount of white in the dorsal coat which usually is of a "variegated" nature, i.e., white hairs are usually interspersed among pigmented hairs giving a kind of roan or silvering effect, which increases in intensity with the amount of white in the fur. ¹

1. The "m(W)" Complex

The amount of white on the dorsum of W/+ heterozygotes is subject to selection. Thus Dunn ( 1937) succeeded in establishing a stock in which the dorsum of W animals displayed, on the average, about 90% white with a typical roan pattern. On the other hand the +/+ segregants of this strain either displayed no white at all, had occasionally small belly spots, or had a very small solid (not variegated) white patch on the center of the back. Moreover, the spotting in these +/+ animals seemed to be due not to the same modifiers which increased the amount of white in the W/+ segregants but rather to some " k" genes (see Chapter 9, Section II, D) which were carried in the stocks and simultaneously selected for.

As a consequence of these modifying genes, ² which seem to behave as almost complete recessives, W can behave either as nearly completely dominant in its effect on the fur, or it can act as almost completely recessive. ³ Indeed, because in a stock homozygous for the normal alleles of the m(W) complex W/+ mice are generally uniformly pigmented, and those W/W (severely anemic) animals which survive long enough to grow fur are black-eyed whites, ⁴ the customary designation of " W" as "dominant spotting" can be doubly misleading; it may act as a recessive and does not always produce a spotted phenotype! As most mouse stocks contain some mutants of the m(W) complex, W usually behaves as a semidominant [and, at least in the C57BL/6 strain, rarely produces dorsal spotting ( E. Russell et al., 1957)]. Furthermore, even when incorporated into an inbred strain W//+ mice show varying degrees of variegation as a consequence of nongenetic factors.

2. Interaction with Piebald (s) and the "k" Complex

So far, we have considered the expression of W and its specific modifiers in the absence of the gene for piebald spotting ( s and the " k" genes. Since the simultaneous occurrence of W and s/s in the same genome has a synergistic effect on the amount of white spotting, W/+;s/s mice are either entirely white with black-eyes, or have small pigmented areas in the region of the ears and/or the haunches. ⁵ In fact, even when s is heterozygous with its normal allele it slightly augments the amount of white spotting in W/+ mice in the presence of m(W) genes.

Insofar as the interactions of m(W) with s and with the " k" genes are concerned the evidence is incomplete. According to Dunn ( 1937) "the indications are that the effects of m(W) do not cumulate with those of s but do to a slight extent with those of " k" when both " k" and m(w) are homozygous. The latter two complexes may have some common genes although it is probable that some of the m(W) genes are independent of and act differently from those of the " k" series, since the latter may themselves initiate reactions without W."

B. Viable Dominant Spotting ( W^v)

The second mutation at this locus, W^v (for viable dominant spotting), was recognized and reported by Little and Cloudman ( 1937). ⁶ When heterozygous with the normal allele W^v produces a mid-ventral spot of variable size on the trunk, and very frequently a small mid-dorsal spot on the head as well (see Grüneberg, 1939). The rest of the coat, unlike in W/+ mice, is diluted ( Plate 3-D). ⁷ While Little and Cloudman found that, like W, this mutation when homozygous produced black-eyed whites [ Plate 3-E; occasionally pigment occurs in the skin of the ear pinna but not in the hair ( Figure 10-1)], ⁸ these homozygotes survived significantly longer than W/W mice. In contrast to W/W genotypes, all of which died within 2 weeks after birth, and the majority of which died earlier — many of them in utero ( deAberle, 1927) — many W^v/W^v mice lived for more than 3 weeks and some lived to be adults. Indeed, Little and Cloudman found a W^v/W^v female and one male to be fertile, although this fertility was temporary and lasted only for a limited time. ⁹

C. Influence of W and W^v on Pigment Granules

Various attributes of the pigment granules in W/+ and W^v/+ mice have been studied in some detail by E. Russell ( 1949c) who found that whereas one dose of W has no effect on hair pigment intensity, one does of W^v has a slight but significant one. Thus on full-color black ( a/a;B/B), chinchilla black ( a/a;B/B;c^ch/c^ch), full-color brown ( a/a;b/b), pink-eyed sepia ( a/a;B/B;p/p) and full-color yellow ( A^y/—) backgrounds, W^v/+ have fewer pigment granules than +/+ animals, a difference which is most apparent in the number of medullary granules. In some of these genotypes the size of the granules also are smaller in W^v/+ than in the corresponding +/+ type. As pointed out by Russell these observations are compatible with the concept that W^v/+ substitution causes a reduction in the general level of pigmentation. ¹⁰

D. Etiology of Spotting in W and W^v Genotypes

All available evidence indicates that the inviability of melanoblasts is responsible for the white spotting of the coat of W-series genotypes. Moreover, this lethality appears to be due to these alleles acting within the melanoblasts themselves rather than via the skin. Evidence that this is the case stems from (1) Mintz's ( 1970) observation that W/W <--> +/+ allophenics are either completely pigmented or white-spotted; ¹¹ (2) grafting results which indicate that the pigmented and nonpigmented areas of the allophenics which are spotted are composed of both W/W and +/+ cells (see Chapter 7, Section VII); and (3) the results of experiments carried out by Mayer and M.C. Green ( 1968) and Mayer ( 1970, 1973a).

Mayer and M.C. Green observed that when neural tube, including neural crest cells, from 9-day-old embryos derived from W^v/+ matings, was combined with putative neural crest-free +/+ skin (originating from the lateral flank between the fore and hind limb buds of 11-day-old embryos) and allowed to differentiate in the coelom of the chick, 61% of the transplants failed to produce pigment. Since this number is significantly greater than the expected number of W^v/W^v combinations, i.e., one would expect 25% of the grafts to include W^v/W^v neural tubes, it seems likely that a considerable number of W^v/+ neural tubes must likewise have failed to give rise to melanocytes (see note 16).

Mayer and Green also found that when neural tubes from 9-day-old +/+ embryos were combined with skin from 11-day-old embryos produced by W^v/+ matings, all the grafts were pigmented. Thus it seems apparent that there are no differences in the ability of skin from +/+, W^v/+, and W^v/W^v embryos to support the differentiation of +/+ melanoblasts, again an observation completely in accord with Mintz's hypothesis.

In further support of these conclusions Mayer ( 1970) obtained similar results when he combined small pieces of 9-day-old +/+ neural crest-containing neural tube with W^v/W^v skin obtained from 13- to 18-day-old embryos, skin whose genotype could at this age be confirmed by the pale color of the donor's liver. When such composite transplants were allowed to incubate for 15 days in the flank of White Leghorn chick embryos, all of them possessed large numbers of pigmented hairs. In fact, in no case were any pigment-free hair follicles observed.

Because Mayer obtained different results when he employed neural crest-containing albino skin, i.e., such skin, presumably because it possessed c/c melanoblasts, prevented the entrance of +/+ melanoblasts (see Chapter 11), he notes that one possible conclusion of his findings is that melanoblasts never enter the skin of W^v/W^v embryos. However, as he also points out, an alternative explanation, and one which seems more likely, is that W^v/W^v melanoblasts enter the skin but die soon after. ¹²

Mayer ( 1973a) also determined the fate of reciprocal combinations of 13-day-old embryonic W/W^{v 13}and +/+ epidermis and dermis when grafted to the chick coelom, and these results too were completely in accord with the fact that there was nothing wrong with the mutant's skin. Thus melanoblasts which were present in either the +/+ dermis or epidermis could move freely into the corresponding W/W^v component, differentiate, and form pigment. ¹⁴

Despite the fact that all the evidence indicates that W-locus alleles act within the melanoblast, Gordon ( 1977) has reported some observations which he believes are difficult to reconcile with Mintz's notion that W/+ mice possess two kinds of melanoblast clones, completely normal and inviable. He made W/+;+/+ <--> +/+;c/c allophenics and found that such animals usually were completely white although a few possessed traces of pigment. Their eyes were ruby colored and microscopic examination showed the eye pigment to be located only in the retinal epithelium which was a mosaic of black and white sectors. It thus appears that W/+ melanoblasts while capable of pigmenting most of the coat (as well as the eyes) of W/+ mice, populated exceedingly few hairs when in competition with +/+;c/c (albino) melanoblasts. Although this is not surprising in the sense that it is known that in some allophenic combinations the melanoblasts of one genotype often have an inherent ability to dominate over those of the other, due at least in part to their greater proliferative capacity, Gordon also found that +/+ cells of similar genetic origin as the W/+ cells frequently pigmented most of the coat of +/+;+/+ <--> +/+;c/c allophenics. It therefore appears that none of the melanoblasts of W/+ mice are as competent as those of coisogenic +/+ animals in competing with albino melanoblasts. Accordingly, Gordon believes that the melanocytes of W/+ mice are inherently weaker than those of +/+ animals (or it could be argued that they possess both inviable and weaker that viable +/+ clones.) ¹⁵ However, there is an alternative explanation for these findings, one which again is in complete accord with Mintz's hypothesis. Thus if one accepts the fact that to begin with W/+;+/+ <--> +/+;c/c allophenics possess, on the average, only half the number of potentially pigmented melanoblast clones as +/+;+/+ <--> +/+;c/c allophenics (and these are outnumbered 2:1 by melanoblast clones of albino origin) it does not seem very surprising that so far all of them have been completely or almost completely unpigmented. This is especially the case since only relatively few of these allophenics have been produced (see note 11). ¹⁶

E. Expression of Extraepidermal Melanocytes in W^v/— Mice

Although all the evidence is consistent with the conclusion that the effect of the W-series of alleles on white spotting of the coat is due entirely to a defective population(s) of melanoblasts, and is completely independent of any direct influence of these genes on the skin itself, this does not rule out the possibility that other tissue environments can influence the capacity of W-mutant melanoblasts to survive and/or form melanin. Indeed, evidence that such is the case is derived from the study of Markert and Silvers ( 1956) who, after surveying the occurrence of melanocytes in the nictitans, harderian gland, hair follicle, ear skin, choroid, and retina of 50 different genotypes (see Table 10-1), concluded that "the capacity of a tissue to elicit melanogenesis depends upon the embryonic history of the tissue (i.e. what kind of tissue it has become — nictitans, harderian gland, etc.) and upon the genetic composition of the tissue." This fact has been reemphasized by two ingenious studies of Deol ( 1971, 1973). In the first of these investigations he analyzed the pigmentation patterns in the choroid, harderian gland, and inner ear in a number of spotted genotypes and concluded that the host tissue plays an important role in determining the pattern of spotting and that all melanoblasts may not be affected to the same degree. For example, he found that in the harderian gland of W^v/+ mice the number of melanoblasts was not only slightly reduced from that observed in +/+ glands but that they were on average smaller than normal, although cells of normal size were quite common. ¹⁷ Deol also observed that although spotting of the choroid was significantly heavier in W^v/+ animals with mid-dorsal head spots than in those without them, the amount of white spotting of the coat was not a reliable guide to internal pigmentation, nor was any general trend evident when the effects of different genes were compared (see Table 12-1).

In his later study, Deol focused his attention on the pigmentation of the eye and compared the pigmentation of the iris with that of the choroid and the retina in the same eye of different white spotted genotypes. This was particularly appropriate since the outer layer of the iris derives its melanocytes from the choroid while the melanocytes of the inner layer of the iris are derived from the retina. Thus, as Deol points out, any pigmentation differences between the choroid and outer layer of the iris or between the retina and inner layer of the iris would constitute evidence that different tissue environments can affect the survival, differentiation, and/or melanogenic capacity of pigment cells. Such differences were found (see Table 12-2). In W^v/+ mice, the choroid was partially pigmented (spotted) while the outer layer of the iris was fully pigmented suggesting that identical melanocytes (from the choroid) were responding independently to these different environments. In W^v/W^v mice, however, both the choroid and outer layer of the iris were devoid of pigment, presumably because the melanoblasts of these animals were so abnormal that they could not differentiate (or survive) in either environment.

F. Influences of W^v on the Inner Ear

The inner ears of all W^v/W^v mice have marked abnormalities in the cochlea (the most striking abnormalities occur in the organ of Corti and the stria vascularis) and many have severe abnormalities in the saccule as well. ¹⁸ These anomalies also occur, though in a more benign form, in a small part of the cochlea of a few old W^v/+ animals ( Deol, 1970c). Since there is good evidence that the neural crest contributes to the formation of the acoustic ganglion, it seems most likely that it is via some neural crest defect that these pathological changes in the inner ear are produced ( Deol, 1970c). These observations, therefore, provide further support for the thesis that the effect which W^v has on coat color likewise results from a direct influence on the neural crest. ¹⁹

G. Influence of W and W^v on Hematopoiesis

There are three coat-color determinants in mice, flexed-tailed, steel, and dominant spotting, all of which produce white spotting and all of which are associated with congenital anemias. The most thoroughly investigated of these anemias is the one associated with the W-series of alleles, especially W and W^v. Indeed, the anemic condition caused by these alleles has been studied so extensively that this aspect of the W-gene action alone could easily form the basis of an impressive monograph. The early efforts on this subject are well reviewed by Grüneberg ( 1952) and by E. Russell ( 1954). A more recent review is included in the second edition of the Biology of the Laboratory Mouse ( E. Russell and Bernstein, 1966) and much of what follows is based on this reference (see also E. Russell, 1970).

As we have already noted animals homozygous for W are characterized by a very severe macrocytic anemia ( Attfield, 1951; E. Russell and Fondal, 1951), so severe in fact that they usually die within a few days after birth. W/W^v and W^v/W^v mice likewise suffer from this condition and although it is not as deleterious in these genotypes it can be lethal in some, especially between birth and weaning when they are growing most rapidly. Animals which survive this period usually live more than a year ( E. Russell and Bernstein, 1966).

While W has no effect on erythropoiesis when heterozygous with the normal allele, the number of erythrocytes is slightly reduced and their mean cell volume slightly increased in W^v/+ genotypes ( Grüneberg, 1942; E. Russell, 1949a). Thus the anemic conditions caused by the alleles W and W^v can be ranked in terms of the number of erythrocytes: +/+ (normal) = W/+ > W^v/+ > W^v/W^v > W/W^v > W/W.

In the severely affected genotypes the anemia already is apparent when the liver succeeds the yolk sac blood islands as the principle site of hematopoiesis at 12.5 days gestation ( Borghese, 1959) and it persists for as long as the animal lives. On a heterogeneous background the mean erythrocyte counts of newborn W/W, W/W^v, and W^v/W^v mice are 0.8 x 10⁶, 1.4 x 10⁶ and 2.2 x 10⁶ RBC/mm³, respectively ( E. Russell and Fondal, 1951), counts which differ significantly from the 4.8 x 10⁶ RBC/mm³ for normals of the same age ( E. Russell, 1954). ²⁰ Although on specially selected genetic backgrounds the postnatal survival of W/W anemics can be prolonged to a mean of 10 days, with an occasional animal surviving to adulthood, under these conditions as well the number of red cells/mm ³ (1.5 - 1.9 x 10⁶) at birth is very low ( E. Russell and Lawson, 1959).

Nevertheless, the anemia in these severely affected genotypes is not aplastic but hypoplastic. The absolute number of erythrocytes shows the same relative increase from the sixteenth day of gestation to birth in W/W mice as it does in +/+ genotypes ( E. Russell and Fondal, 1951), and the proportion of reticulocytes in the blood of W^v/W^v adults ( Niece et al., 1963) and of W/W newborns ( deAberle, 1927) is higher than in their +/+ littermates. The fetal liver and neonatal marrow of W/W, W/W^v, and W^v/W^v animals is hypoplastic ( E. Russell, 1954; Borghese, 1959), while the marrow cellularity of adult W/W^v and W^v/W^v mice is nearly normal ( E. Russell et al., 1953). All the evidence indicates also that there is nothing wrong with the life span of the erythrocyte ( Niece et al., 1963). ²¹

There is good evidence that the anemia produced by the W-alleles results from a delay in the maturation of erthrocytes ( E. Russell et al., 1953; Borghese, 1959; Benestad et al., 1975; Shaklai and Tavassoli, 1978). When isotopically labeled heme-precursors were innoculated into W/W^v mice and their +/+ littermates, erythrocytes with labeled protoporphyrin appeared in the circulation of the normal mice after 3 days but not until 7 - 14 days in the anemics ( Altman and E. Russell, 1964). This delay in the capacity of W/W^v mice to form erythrocytes, a delay which seems to be caused by a deficiency in the number or differentiating capacity of erythropoietic stem cells ( McCulloch et al., 1964; M. Bennett et al., 1968a), is responsible also for their great sensitivity to X-irradiation ( Bernstein, 1962; E. Russell et al., 1963).

That this defect in the maturation of erythrocytes results from W-alleles acting directly upon the blood-forming tissues, and not, as in the case of steel (see Chapter 11, Section I, E), indirectly by affecting the microenvironment in which hematopoiesis occurs, is demonstrated by the fact that W/W, W/W^v, and W^v/W^v anemic animals can be cured completely and permanently by implantation of histocompatible (syngeneic) +/+ blood-forming tissue from adult marrow or from fetal livers (E. Russell et al., 1956b, 1959; Bernstein and E. Russell, 1959; Bernstein, 1963; E. Russell and Bernstein, 1968), or even from the placenta ( Dancis et al., 1977). Indeed, this anemia can also be cured by intravenous administration of histoincompatible (allogenic) hemopoietic cells from either very compatible donors ( E. Russell and Bernstein, 1967; Harrison, 1972a; Harrison and Cherry, 1975) or into immunologically tolerant or immunosuppressed hosts (Seller 1966, 1967, 1968, 1970, 1973; Seller and Polani, 1966, 1969). ²²

H. Influence of W and W^v on Gametogenesis

In addition to their effect on coat color and hematopoiesis the alleles of the W-series influence the development of the primordial germ cells. W/W^v mice always are sterile and, on most genetic backgrounds, W^v/W^v mice are too ( Grüneberg, 1952; Veneroni and Bianchi, 1957). ²³

A histological study of the gonads of W/W, W/W^v, and W^v/W^v mice, and their normal counterparts, from 0 to 28 days postpartum revealed a drastic deficiency in the number of germ cells in the potentially sterile gonads of both sexes ( Coulombre and E. Russell, 1954). The severity of this defect seemed to parallel that of the anemia, i.e., it was most severe in W/W animals and more severe in the W/W^v than in W^v/W^v genotypes. This impairment seemed to reflect both a reduction in the number of definitive germ cells and a retardation of their maturation. Thus, in contrast to the situation in the normal (+/+) ovary which contains many large follicles at 1 month, corpora lutea by 6 weeks, and continues to produce large numbers of ova which develop normally long after 1 year ( E. Russell, 1954), the ovaries of W^v/W^v females possess very few ovarian follicles at any time and these cease to grow and develop after the age of 2 months ( E. Russell and Fekete, 1958). ²⁴ The adult W^v/W^v testis too is abnormal, being almost devoid of spermatogenesis. Most of the tubules contain only Sertoli cells, and the few germ cells which occur are almost all spermatogonia ( Coulombre and E. Russell, 1954; E. Russell, 1954).

1. Embryological Basis of Germ Cell Defect

It follows that if the influence which these W-alleles have on gametogenesis is a consequence of their effect on erythropoiesis then this latter effect must precede the germ cell anomaly. To determine if this was the case Borghese ( 1956) explanted gonads from 12-day-old W/W and +/+ fetuses into a favorable tissue culture environment. Because these gonads continued to develop as they would have if left in situ, Borghese concluded that the sterility was independent of the anemia. Similar observations were made by E. Russell and her associates ( 1956a) who transplanted W/W and W^v/W^v gonads of the same age to the spleens of histocompatible +/+ hosts. Nevertheless, while these observations indicated that the germ cell defect had reached full expression at the earliest stage (12.5 days) at which evidence of the erythropoietic defect had been identified, they did not prove that the germ cell defect preceded the anemia. These experiments could just as easily be interpreted as a simple failure to revive germ cell formation in an already deficient gonad.

The best evidence that the germ cell defect is independent of the anemia stems from the elegant analysis of Mintz and E. Russell ( 1957). By elective staining of primordial germ cells of embryos derived from W/+ and W^v/+ matings with the azo dye coupling technique for alkaline phosphatase, they were able to follow the migration of germ cells from their place of origin, the yolk sac splanchnopleure, to their definitive positions in the paired germinal ridges. They found, in agreement with the observations of Chiquoine ( 1954), that in normal embryos the migration of these cells occurs between the eighth and twelfth days of embryonic life and that this migration is accompanied by their continued multiplication. ²⁵ Thus during this 4-day interval the number of germ cells in +/+ mice was found to increase from a maximum of 76 to a maximum of 5711 ( Mintz and E. Russell, 1957). On the other hand, while W/W, W/W^v and W^v/W^v embryos possessed normal numbers of primordial germ cells at 8 days of age, the number of these cells failed to increase thereafter and were retarded in their migration to the germinal ridges. By day 12 these affected genotypes possessed as few as 18 and a maximum of 72 germ cells. ²⁶

It is therefore apparent that the defect in gametogenesis in W/W, W/W^v and W^v/W^v mice is expressed as a mitotic failure, evident at 9 days, and it is very likely that the cause of this failure is present before this time. Consequently it is very unlikely that the sterility is a secondary result of the anemia. Indeed, even if an undetected defect in yolk sac hematopoiesis should occur at 8 days, i.e., earlier than the observed germ cell anomaly, it is most unlikely that it could physiologically affect the activities of the germ cells because of the absence of a functional embryonic circulation at this early stage of development ( Mintz, 1957a). It therefore seems reasonable to conclude that the pleiotropic effects of W-series alleles "stem from a single gene-mediated alteration to which certain kinds of cells are peculiarly vulnerable because of their own special activities" ( Mintz, 1957a). ²⁷

I. Other Alleles

In addition to W and W^v the W-locus is represented by the following alleles:

1. Ames Dominant Spotting ( W^a)

There is not much information on this allele. It was found among the offspring of an X-rayed male of the Z strain and resembles W except that W^a/+ heterozygotes have a prominent head blaze. Homozygotes are anemic and die within a few days after birth. W/W^a young are also anemic ( Hollander, 1956; Schaible, 1963a). Schaible ( 1969) employed this W-allele in his studies on white spotting and notes that when heterozygous (with +) W^a not only causes spotting but dilutes black pigment to some shade of grey. He also states that some animals "may show variegation in that one or more patches will be of a color different from the expected shade of grey." ²⁸

2. Ballantyne's Spotting ( W^b)

This W-series allele occurred in the C57/St strain at Roswell Park. Its characteristics have been described by Ballantyne (from whom it gets its name) and his associates ( 1961) and what follows is based on their description. The original mutant was a female who showed "an extensive diffuse-white-spotting of the back and sides with a small frontal blaze." The belly was predominantly white and the pigmented areas on the dorsum were significantly lighter in color than the intense black characteristic of C57/St mice. When hairs were plucked from these various regions and examined microscopically it was found that they could be classified as follows: (1) completely white; (2) pigmented at the tip but nonpigmented for variable lengths basally; (3) pigmented at the tip and irregularly pigmented thereafter; and (4) relatively uniformly pigmented from tip to base. The "basal dilution" of pigmentation observed in many hairs appeared to be related to a striking reduction in medullary granules.

Breeding studies revealed that, like W, W^v, and W^a, W^b is inherited as a semidominant. W^b/W^b genotypes are black-eyed white and presumably anemic since they are pale at birth with a median life span of 8 days. All W^b homozygotes which survive are sterile. Test matings with W^v, and with another putatively unique W-allele called W^s, have confirmed its W-locus assignment. ²⁹

Although W^b/+ mice more closely resemble W^v/+ than W/+ animals, they usually are more extensively spotted than W^v/+ and microscopic examination of pigmented hairs from W^v/+ mice failed to reveal the extensive terminal deficiency of medullary pigmentation regularly found in W^b/+ hairs ( Ballantyne et al., 1961). ³⁰

3. W-Fertile ( W^f)

This mutation has recently been described by Guénet and his associates ( 1979) and the following is based entirely on their account (see also Guénet and Mercier-Balaz, 1975).

The first animals carrying this mutation were discovered when in the course of producing a W^v/+ C3H/He congenic line, one C3H/He female produced two black-eyed white males (presumably W^v/W^f heterozygotes) with greyish patches at the roots of the ears. Both of these mice looked healthy, fared well in competition with their littermates, and subsequently proved fertile.

W^f/+ animals on a C3H/He background are characterized by sharply demarcated white spots on the forehead and belly. They also have a white tail tip ( Figure 10-2). The spots vary in size, sometimes consisting of only a few white hairs. The coat itself is not diluted as in W^v/+ heterozygotes. When transferred onto other isogenic backgrounds, or when expressed in F₁ hybrids (e.g., with C57BL/6 or 129Sv) the phenotypic expression of W^f may completely vanish so that W^f/+ heterozygotes are indistinguishable from +/+ animals ( Figure 10-2), a situation which suggests that modifier genes, perhaps similar to those in the m(W) complex, are responsible for the expression of this allele when heterozygous with +.

Both W^f/+ and W^f/W^f males and females are fertile. Between 1 and 6 weeks of age W^f homozygotes are less viable than their W^f/+ or +/+ littermates and some become "runted" and die after 1 or 2 weeks of age. Deaths during adulthood however are rare.

As is the case with other W-alleles, W^f interacts with several other spotting genes. Thus, in compounds with rump-white ( Rw) (see Section II, C), i.e., Rw+/+W^f, exhibit spotting patterns analogous to those of Rw/+ mice except that the white area extends up to the belt. When combined with steel-Dickie ( SI^d) (see Chapter 11, Section I, C, 1) ( Sl^d/+;W^f/+) a phenotype is produced which looks very much like Sl^d/+;W^v/+. Pigmented hairs are scattered among the unpigmented ones over most of the body without a definite pattern and the ventrum is almost invariably white. Finally, W^f/+;s^l/s^l (piebald lethal; see Chapter 9, Section II, B) compounds are almost completely white with the exception of some relatively intensely pigmented areas on the shoulders or haunches.

In addition to its influence on pigmentation, W^f also produces a chronic macrocytic anemia. Thus Guénet and his associates found adult +/+, W^f/+, and W^f/W^f mice to have red cell counts (x 10⁶/mm³) of 8.00 +/- 0.10, 7.29 +/- 0.12, and 6.29 +/- 0.19, respectively. The mean cell volumes of their erythrocytes (microns³) were 46.55 +/- 0.73 (+/+), 49.25 +/- 0.53 ( W^f/+), and 54.47 +/- 1.13 ( W^f/W^f). These results, therefore, are consistent with data concerning other W-alleles. Moreover, these investigators have also found that the bone marrows of W^f/+ and W^f/W^f mice do not possess a normal number of cells capable of forming macroscopic colonies in spleens of irradiated coisogenic recipients, a finding also in accord with those reported for other W-locus genotypes ( E. Russell, 1970).

Clearly the most interesting feature of this allele is that, unlike the others, when homozygous it neither produces an all-white phenotype nor does it appear to have any demonstrable effect on gametogenesis. This occurs despite the fact that such homozygotes are anemic. Although it is conceivable that further study will reveal some influence on gametogenesis, nevertheless, taken together, this mutation provides further evidence that there is no direct relationship between the triad of effects associated with W-series alleles.

4. Jay's Dominant Spotting ( W^j)

This W-locus allele was found by George E. Jay in his C3H colony at the National Institutes of Health. It has been analyzed by E. Russell and her colleagues ( 1957) and the following is based largely on their findings. When heterozygous with the normal allele W^j produces extensive white spotting on the ventrum with some white on the back especially on the crown of the head. There is no apparent diminution of pigment intensity in the colored areas of the coat in W^j/+ heterozygotes nor are they anemic. When homozygous W^j produces black-eyed whites with an average survival of about 8 days (0 - 18). These animals are severely anemic with blood counts averaging 1.04 (+/- 0.04) x 10⁶ RBC/mm³ at 0 - 1 day of age [as compared with a mean of 4.58 (+/- 0.24) x 10⁶ TRBC/mm³ for their normal littermates]. They also are deficient in germ cells, a deficiency which has been analyzed embryologically by Mintz ( 1957a) and which is indistinguishable from her observations for W and W^v ( Mintz and Russell, 1957).

Test crosses with both W/+ and W^v/+, as well as with Sl/+, have confirmed that W^j is a member of the W-series, a member which appears to resemble W in all respects except for the increased amount of white spotting, especially on the ventrum. ³¹

5. Panda-White ( W^pw)

This spontaneous mutation occurred at Oak Ridge. In heterozygotes ( W^pw) the pigmentation of the coat is restricted mainly to the head (the snout, near they eyes, and at the base of the ears) and the base of the tail, but sometimes small pigmented patches occur on the hips and shoulders as well. Homozygotes are severely anemic and die within 3 days postpartum. W^pw/W^v heterozygotes are black-eyed whites and sterile ( Steele, 1974).

6. Sash ( W^sh)

This putative W-allele was recently reported by Lyon and Glenister ( 1978). It occurred spontaneously at Harwell in a pair set up to provide (C3H x 101)F₁ hybrid stock. The original mutant has a broad sash of white around its body in the lumbar region and produced offspring like itself when bred to a normal animal. When crossed to mice carrying patch ( Ph) or rump-white ( Rw), the double heterozygotes showed an additive interaction in their spotting pattern, but heterozygotes with W^v were black-eyed whites, with small patches of pigment around the ears and eyes. All three types of heterozygotes proved fertile, and no crossing-over between sash and Ph, Rw, or W has yet been observed in several hundred offspring. Since sash displays a nonadditive interaction with W, it is considered to be an allele of W and has been given the symbol W^sh.

Originally, sash appeared to be lethal when homozygous. However, in crosses of W^sh/+ x W^sh about 1% of the offspring were black-eyed whites. These black-eyed whites proved to be heterozygous for the original lethal sash and a new viable type. Homozygotes for the viable sash are black-eyed whites, viable and fertile. The interpretation is that the original sash carried a linked recessive lethal, and that in the viable type this lethal was lost by crossing-over. The data indicate that the lethal occurs about 1 cM from W, but which side is not yet known.

No evidence of anemia has been found in W^sh/+, W^sh/W^sh, or W^sh/W^v genotypes. Thus W^sh is an unusual allele in that it shows the full effects of the W-locus on spotting, but appears to have no effect on erythropoiesis or gametogenesis.

7. Extreme Dominant Spotting ( W^e)

This W-allele also has only recently been described ( Cattanach, 1978). It occurred at Harwell in a Rb(5.15)3Bnr stock maintained on a C3H/H-101/H genetic background. It closely resembles Ballantyne's spotting ( W^b) in that when heterozygous (with +) it produces a white belly spot with extensive white markings on the head and body, and a general lightening of the coat. Homozygotes are anemic black-eyed whites which, so far, have not survived beyond 10 days. Compounds with W^v are also black-eyed whites but survive to maturity and are sterile. Compounds with rump-white ( Rw) are viable and fertile black-eyed whites with some very limited pigmentation in the ear skin. This new W-mutation has been given the provisional name of extreme dominant spotting ( W^e).

8. Other Alleles

In addition to the above, Edwin Geissler and E.S. Russell ( 1978) currently are analyzing 10 putative W-alleles all of which occurred spontaneously in the C57BL/6J strain at the Jackson Laboratory (see also E. Russell and Bernstein, 1974). Since these mutations are on the same genetic background they are especially advantageous for comparing their pleiotropic effects and in many cases the severity of these effects in different tissues does not correlate at all well. For example, while seven of these alleles produce severe anemic conditions when homozygous — so severe that only 0-11% of animals homozygous for these alleles are viable at birth and all succumb shortly thereafter — the amount of spotting associated with these same alleles when heterozygous (with +) varies from as little as 4% (limited to the ventrum) to as much as 95%. Moreover, some of these heterozygotes display a severe pigmentary dilution while in others this manifestation may not be present at all. These observations are especially interesting because they provide further evidence that the influence which the W-series of alleles have on hematopoiesis, gametogenesis, and melanoblast survival are unrelated. Indeed, one wonders if the W-locus is after all a functional unit, or a complex of separate regions for different functions (E. Russell, personal communication)?

The effects of some of the W-locus genotypes considered above are summarized in Table 10-2.