Chapter 10

Dominant Spotting, Patch, and Rump-White

II. Patch ( Ph) and Rump-White ( Rw)

Although patch ( Ph) and rump-white ( Rw) will for the most part be considered separately, they are introduced together because as noted above they are very closely linked to each other as well as to the W-locus on chromosome 5. In fact, these three loci are so closely associated that it seems logical at present to regard them "as a gene triplet which has arisen by repeated duplication of the original chromosome segment" ( Searle and Truslove, 1970). The upper fiducial limits (P=0.05) for map distances are 0.08 cM for Ph and W^v, 1.4 cM for Ph and Rw, and 0.4 cM for Rw and W^v ( Grüneberg and Truslove, 1960; Searle and Truslove, 1970). ³²

A. Patch ( Ph)

Almost all the information on this mutant stems from a detailed analysis by Grüneberg and Truslove ( 1960) and what follows is essentially a summary of their findings.

Although patch arose as a spontaneous mutation in the C57BL strain, Grüneberg and Truslove's study was conducted on an outcross to CBA/Gr.

1. Spotting Patterns of Heterozygotes

White spotting in Ph/+ animals is like the spotting in piebald ( s/s) and belted ( bt/bt) mice in that the areas of white fur usually are sharply demarcated from the pigmented areas and only occasionally does roan or variegated spotting, i.e., the intermixture of white and pigmented hairs as is characteristic of dominant spotting ( W/+ and W^v/+), occur. While Ph/+ mice always display at least a large belly-spot as well as extensive tail spotting and white digits, and there is frequently a large white patch in the middle of the trunk, the amount of white spotting is highly variable (Figures 10-3 and 10-4a). Moreover, as with other spotting genes, this variability depends upon the genetic background. Thus when Grüneberg and Truslove outcrossed their patch stock to three different inbred strains (A, C57, and CBA) the F₁ Ph/+ mice displayed widely different amounts of dorsal white. The spotting pattern itself seems to result from "the gradual spread of a belly-spot to form a belt (sometimes with a secondary belt in the shoulder region) which then expands first posteriorly and then anteriorly."

2. Interactions with Other Spotting Determinants

When tests were carried out to establish the relationship between patch and some other spotting genes it was found that they interacted synergistically. Thus when Ph/+;s/+ mice were backcrossed to +/+;s/s animals the Ph/+;s/s offspring resembled the black-eyed white ( W/+;s/s) of the Mouse Fancy, being either completely white, or with small pigmented patches round the eyes, ears, or on the haunches ( Grüneberg and Truslove, 1960). Patch likewise interacts with belted ( bt), white ( Mi^wh), and viable dominant spotting ( W^v) ( Grüneberg and Truslove, 1960) as well as with dominant spotting ( W), steel ( Sl) ( Wolfe and Coleman, 1966), steel-Dickie ( Sl^d), and rump-white ( Rw) ( Deol, 1970b; Searle and Truslove, 1970). Patch, belted ( Ph/+;bt/bt) mice usually have pigmented heads and shoulders while the rest of the body is either completely white or has a little pigment on the haunches. Patch, white ( Ph/+;Mi^wh/+) mice resemble Ph/+;bt/bt animals in that pigment is confined to the head, shoulders, and forelegs, with sometimes a little on the haunches. However, unlike Ph/+;bt/bt animals, the pigmented areas of these mice are dilute as a consequence of the effect of Mi^wh/+ (see Chapter 12, Section I, B, 1). In the double heterozygote of patch and rump-white ( Ph+/+Rw) the head (except for a small spot) and the shoulder region are almost fully pigmented, while the remainder of the body is white although occasional pigment patches occur ( Figure 10-4b). The same description applies to the patch and steel-Dickie ( Ph/+;Sl^d/+) double heterozygote, except that in these the pigmented fur is diluted ( Figure 10-4c).

The most profound effect on pigmentation is found when patch is combined with W^v ( Figure 10-5). Ph+/+W^v mice are completely unpigmented except for their head which is usually pigmented as in Figure 10-4d but is sometimes pigmented only on the cheeks and ears. Moreover, even the pigmented areas are diluted to a greater extent than can be accounted for solely by the influence of W^v/+. These animals also have less pigment in the inner ear than either heterozygote alone, a deficiency associated with more severe inner ear abnormalities (see Deol, 1970b).

3. Other Effects

The only other anomaly found in Ph/+ mice is that their skull is a little wider and shorter than normal and has a large interfrontal bone ( Grüneberg and Truslove, 1960).

4. Viability of Homozygotes

Inasmuch as all patch mice tested have proved to be heterozygous and since matings between such animals produce a 2:1 rather than a 3:1 ratio of spotted to self mice, it is evident that the Ph/Ph homozygote dies sometime before birth. To determine when, Grüneberg and Truslove examined embryos from both F₂ ( Ph/+ x Ph/+) and backcross ( Ph/+ x +/+) matings at various stages in their development. They found in the F₂ matings (but not in the backcrosses) that some late embryos (12-17 days old) were severely disfigured by a condition that they designated as "cleft face." Nevertheless, the number of these "monsters," which obviously were Ph/Ph homozygotes, only accounted for about a third of their expected number. Clearly, about two-thirds of the Ph/Ph embryos had died before they reached this stage.

Further studies revealed that the primary abnormality of Ph homozygotes is an increase in their water content (hydrops) which seems to develop between the 8- and 9-day stage. ³³ Abnormal accumulations of liquid are observed first flanking the notochord posteriorly and later anteriorly. To a varying extent, excess fluid also occurs in the circulation, pericardium, under the epidermis, and within the tissues. The more severely affected embryos die around the tenth day but about one-third survive longer. These subsequently develop a subepidermal bleb in the middle of the face which interferes mechanically with the formative movements of the nose and palate and hence is responsible for the "cleft-face."

It appears obvious from the more extreme manifestations of the Ph homozygous condition that the total water content of the embryo is greatly increased and that this excess fluid must be of external origin. Whether it enters via the allantois, or whether it is derived from the amniotic cavity of from the extraembryonic coelom is, however, not known.

5. Pigmentation of Homozygotes

The pigmentation of Ph homozygotes has recently been investigated by Mayer (unpublished) who transplanted skin from various regions of two 14-day-old Ph/Ph embryos, identified by "cleft-face," to the testes of compatible hosts. He found that whereas skin from the vibrissae region always produced pigmented hairs, and skin from the shoulder and hip regions gave rise to either pigmented, unpigmented, or a mixture of pigmented and unpigmented hairs, all grafts from the flank region were unpigmented. It thus appears that, unlike splotch ( Sp) homozygotes (see Chapter 11, Section III, A, 2), Ph/Ph animals have some viable melanoblast clones. They also possess pigmented eyes.

6. Influence on Erythropoiesis and Gametogenesis

Because the Ph-locus is so closely linked to the W-locus, Grüneberg and Truslove determined whether Ph influenced erythropoiesis or germ cell development. They found that 13- to 14-day-old Ph/+ heterozygotes were not anemic (see also Searle and Truslove, 1970) and that Ph homozygotes appeared to have the normal numbers of primordial germ cells at 10 days gestation. ³⁴ Whether these homozygotes are, or would be, anemic was not possible to determine but the did find that Ph+/+W^v animals suffered from a slightly more severe macrocytic anemia than that associated with W^v ( W^v/+) alone. Thus while these data do not provide a strong case for the existence of a specific (allelic) interaction between the Ph and W loci, neither do they rule it out ( Grüneberg and Truslove, 1960).

B. Patch-Extended ( Ph^e)

Recently a second allele has been reported at the patch locus ( Truslove, 1977). It has been given the name of patch-extended ( Ph^e) because only the head and shoulder region of Ph^e/+ animals are pigmented ( Figure 10-6a). ³⁵

When Ph^e/+ mice are crossed with W^v/+ animals the double heterozygote ( Ph^e+/+ W^v) is a black-eyed white ( Figure 10-6b), phenotypically indistinguishable from W^v/W^v. Moreover, these animals look slightly anemic at birth and, unlike Ph+/+ W^v and Rw +/+ W^v, may be sterile as so far none of them (three males and three females) has bred ( Truslove, 1977).

Ph^e +/+ Rw mice look like Ph +/+ W^v animals as they have diluted fur restricted to the sides of the face and ears ( Figure 10-6c).

Although, as in the case of Ph homozygotes, both Ph^e homozygotes and Ph/Ph^e heterozygotes usually die early in gestation, one presumed Ph/Ph^e and one presumed Ph^e/Ph^e embryo with "cleft-face" have been observed. The latter homozygote survived to birth ( Truslove, 1977).

C. Rump-White ( Rw)

This mutation originated in the course of an experiment in which hybrid mice were exposed to fast neutron irradiation ( Batchelor et al., 1966). It has been analyzed by Searle and Truslove ( 1970) and the following is based on their observations.

1. Spotting Patterns of Heterozygotes

Mice heterozygous for rump-white ( Rw/+) usually "have white tails, apart from frequent distal pigmentation, ³⁶ white hind legs and a variable area of depigmentation in the sacral and lumbar regions. This tends to be rather more extensive ventrally than dorsally. Occasionally there are islands of pigmented hair surrounded by white. Digits of forefeet also tend to be white." Three typical Rw/+ mice are shown in Figure 10-7 (see also Figure10-8a).

As in Ph/+ mice, the pigmented areas of the coat of Rw/+ animals show no signs of dilution. In fact, rump-white shows a definite phenotypic resemblance to patch, except that the main area which is unpigmented is in the lumbosacral instead of the thoracolumbar region. The average amount of tail pigmentation also is greater in patch. ³⁷

2. Homozygotes

Rump-white like patch behaves like a fully penetrant dominant mutation which is lethal when homozygous. Although the exact cause of death is not known, dissections of pregnant females from Rw/+ x Rw/+ matings revealed an excess of dead embryos in mid-pregnancy, at about the frequency expected if the comprised the Rw/Rw class ( Searle and Truslove, 1970).

3. Interaction with Other Spotting Determinants

Rump-white also interacts synergistically with other white spotting genes. We have already described the Rw +/+ W^f and the Rw +/+ Ph ( Figure 10-4b) phenotypes. In Rw +/+ W^v mice (Figures 10-8b and 10-9) pigmented hairs are scattered among unpigmented ones in thoracic and lumbar regions as well as on the head. Moreover, there is always a large mid-dorsal head spot and very little pigmentation ventrally. The ears are always pigmented but, as in Ph +/+ W^v animals, there is less pigment in the inner ear (and more inner ear abnormalities) than in either Rw/+ or W^v/+ mice ( Deol, 1970b).

In general, the interaction of Rw with other spotting genes results in the posterior part of the coat being affected more than the anterior ( Searle and Truslove, 1970). Thus rump-white, piebald ( Rw/+;s/s) mice have pigmented hairs on their head and shoulders, but little elsewhere. With lethal spotting ( ls), Rw displays a greater reduction of pigmentation than with s, so that only one or a very few colored patches, mainly dorsal and anterior to the sacrocaudal region, remain ( Searle and Truslove, 1070). Typical examples of the spotting patterns displayed by rump-white, belted ( Rw/+;bt/bt), rump-white, steel-Dickie ( Rw/+;Sl^d/+), and rump-white, splotch ( Rw/+;Sp/+) mice are shown in Figure 10-8c, d and e. With bt, as with s, the area without pigmentation seems less than Grüneberg and Truslove ( 1960) reported for Ph. Furthermore, a similar situation seems to prevail when Rw/+;Mi^wh/+ mice are compared with Ph/+;Mi^wh/+ animals; although both of these genotypes possess pigmented heads, shoulders, and forelegs, only in the former is there considerable pigment in the thoracic region.

4. Influence on Erythropoiesis

Because of the close linkage between Rw and W the blood picture of Rw/+ mice and of Rw +/+ W^v animals has also received attention. As in the case of Ph/+, Rw/+ mice have a normal blood picture while the double heterozygote of Rw and W^v is slightly more affected than W^v/+ alone both with respect to the number of erythrocytes, and their mean corpuscular volume ( Searle and Truslove, 1970).

D. Etiology of White Spotting in Rw/+ and Ph/+ Mice

Although, as in the above testifies, there are obvious differences in the pigmentation patterns of Rw/+ and Ph/+ mice, the most interesting difference was found when thin slices of 1- to 3-day-old skin from various pigmented and nonpigmented regions of these genotypes were cleared, prepared for microscopic examination, and compared. Whereas it was found that in 1-day-old Rw/+ mice dendritic melanocytes were scattered throughout the epidermis, even in areas which were devoid of pigmented hair bulbs, in Ph/+ animals very few pigmented epidermal melanocytes were seen either in regions of pigmented or of white hair. Thus, in the pigmented rump region of Ph/+ mice there were decidedly fewer melanocytes in the epidermis than were found in normal littermates or in the white rump area of Rw/+ mice ( Searle and Truslove, 1970).

The presence of many extrafollicular melanocytes in the white rump region of Rw/+ infant mice indicates that the absence of hair pigment is not due to a failure of melanoblasts to migrate into or survive in this region. Moreover, as pointed out by Searle and Truslove, this is reinforced by the fact that the white spotting of the scrotum and of the tail of these mice includes not only the hairs, i.e., the skin through which these hairs emerge is pigmented (see Figure 10-7). While these observations could be taken as evidence that the white spotting of Rw, unlike that of Ph, is due to some genetic defect which prevents melanoblasts from either entering or maturing in the hairs of the affected region, as has been claimed for belted ( Mayer and Maltby, 1964) ( Chapter 9, Section IV, B), there is an alternative explanation. If one assumes that the melanocyte population in the white rump area of Rw/+ mice represents a secondary population which migrated in after the original population failed to survive because it originated from a defective clone(s) ( Mintz, 1969a), the unpigmented hairs in this region could result from the fact that this "new" population entered the region too late to become incorporated into the developing hair bulbs. Similarly, the white spotting in Ph/+ mice also could be due to the preprogrammed death of specific clones of melanoblasts but at a time too late for them to be replaced by viable cells from adjacent regions. In accord with this interpretation is the observation that whereas in Ph/+ mice there is a sharp demarcation line between the areas of pigmented and nonpigmented hair bulbs ( Grüneberg and Truslove, 1960), in Rw/+ animals unpigmented hair bulbs are sometimes surrounded by pigmented ones and vice versa ( Searle and Truslove, 1970). ³⁸