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| For the s allele: | ||
| s Allele (MGI) | Gene (MGI) | All Alleles (MGI) |
Piebald spotting (s) and its associated "k" complex has been covered in some detail by Grüneberg ( 1952) and much of what follows is based on his excellent account.
Although few mutations have been described at this locus on chromosome 14, it is very likely that some piebalds in existing stocks are of independent origin. This possibility stems from the fact that piebald spotting is carried in many fancier's stocks, and has evidently occurred both in European laboratory mice and in stocks of Japanese waltzing mice. It has been isolated from wild populations in Turkey ( Keeler, 1933), Germany ( K. Zimmerman, 1941), and the United States ( Littleford, 1946).
The most common allele at this locus, s, when homozygous 2 usually produces distinct white spots, i.e., unpigmented and pigmented areas are clearly defined by sharp borders, without any intermingling of white and pigmented hairs ( Plate 2-E). In general, the ventrum displays more white than the dorsum and according to Grüneberg "'centers of depigmentation' are the feet, the tail, particularly its distal part, an area round the umbilicus on the belly, a blaze between the eyes, and the tip of the nose." White shoulder spots may also occur and unite to form a "collar." Lumbar spots likewise often join to form a "belt." As a consequence of modifying factors, known as the "k" complex (see Section D), the extent of all these centers is highly variable, and as they increase in size, they merge in numerous ways. The last areas to lose their pigmentation are the region of the eyes, cheeks, ears, and haunches. Thus s/s may produce entirely or almost entirely unpigmented phenotypes, or it may produce only a few white spots on the belly, feet, and forehead ( Dunn, 1920b).
The amount of "internal" spotting may likewise vary and reflects to some extent the amount of external spotting. Thus whereas a predominantly white a/a;s/s stock described by Mayer ( 1965) and two almost completely white W/+;s/s genotypes examined by Markert and Silvers ( 1956) did not possess any melanocytes in the choroid 3 and harderian gland, almost normal numbers of melanocytes, with only an occasional white spot, occurred in the choroid, and normal numbers of melanocytes were observed in the harderian gland of predominantly pigmented s/s, a/a;s/s, Ay/a;s/s, a/a;b/b;s/s, and Ay/a;b/b;s/s animals ( Markert and Silvers, 1956) (see Table 10-1). Deol ( 1971) reported that in his heavily spotted s/s stock there was a variable degree of spotting of the choroid even though, on average, the unpigmented and pigmented areas were about equal in extent. 4 Moreover, he also observed a great deal of variability in the amount of pigment in the harderian glands of these animals. In the majority of cases this gland seemed to be totally unpigmented but in some the number of melanocytes was severely reduced with the remaining cells unevenly distributed and considerably smaller and less dendritic than normal. Deol also examined 20 s/s ears and observed that one was "lacking in pigment in the region of the lateral crista, another two in the region of the posterior crista, and one in the regions of all three cristae." (Deol's observations on the pigmentation of s/s mice are summarized in Tables 12-1 and 12-2.)
In addition to its effects on pigmentation piebald spotting is also associated with megacolon. Thus Bielschowsky and Schofield ( 1960, 1962) reported that about 10% of their s/s animals had this condition. Since this same affliction is invariably associated with piebald-lethal ( sl) it is considered below.
| For the sl allele: | ||
| sl Allele (MGI) | Gene (MGI) | All Alleles (MGI) |
This allele has been described by Lane ( 1962, 1966). It was observed first in the F2 of a cross between a C3H/HeJ and C57BL/6J. It behaves as completely recessive to wild type and nearly recessive to s. Homozygotes are black-eyed whites with occasional small patches of pigmented hairs on the head and/or rump ( Figure 9-2). Some sl homozygotes die as early a 1 or 2 days after birth while others survive for more than a year. The usual age at death is about 15 days but regardless of how long they live, they all eventually suffer from and usually succumb to megacolon. 5
According to Lane ( 1966) megacolon can readily be distinguished in weaning-age or older animals at autopsy by a markedly distended colon that is filled with hard fecal matter, usually extending from the caecum to varying distances above the rectum. Very young (2- to 15-day-old) mice may or may not show obvious signs of the condition but when they do "their colons are distended and full of soft gelatinous fecal matter in which the beaded appearance of early pellet formation is not present as it is in normal mice of this age."
Since Bielschowsky and Schofield ( 1960, 1962) found that megacolon in s/s mice was associated with a marked reduction of myenteric ganglion cells, Lane determined whether these cells also were deficient in the colons of young sl homozygotes. She found that whereas in 2- to 12-day-old normal mice groups of myenteric ganglion cells of varying size were present approximately every 50-100 microns from the rectum to the caecum, in sl/sl genotypes these cells were lacking at the very distal end of the colon but increased gradually and were present in normal numbers at the proximal end.
Deol ( 1967, 1970b) has reported that the inner ear of sl homozygotes lacks melanocytes and displays severe abnormalities; the cochlear duct is never normal.
| For the s series: | |
| Gene (MGI) | All Alleles (MGI) |
Besides s and sl one or two other possible piebald alleles have been described. One deviant mentioned by Keeler ( 1935) was designated "Berlin-blaze." It was exemplified by very little white and was believed to represent the activity of a different allele with slighter effects. However, it is just as likely that this condition was due to the ordinary s allele's behavior on a unique genetic background ( Grüneberg, 1952).
Another allele, also called sl, was described by Pullig ( 1949). It differed from the usual s allele in that it produced a spotting pattern which was like "a band around the ears extending in varying degrees down the ventral side and looking much like a white scarf tied around the head with free ends of varying lengths." However, as pointed out by Grüneberg, since this type appeared as a mutation in an experiment in which s was also present, and since s and its characteristic pattern were eliminated by "rigid selection," it is not easy to determine if the new spotting pattern was wholly a property of sl, or whether it was to some extent conditioned by the selected genetic background. 6
As already noted stocks of s/s mice may differ widely with regard to the amount of white areas in their coats. Thus by selection and inbreeding it is possible to establish reasonably stable lines of piebald animals which are either essentially all white with very few pigmented areas (hereafter referred to as the "all-white" line), or, have very little spotting, amounting to 10% or less of the coat ( Dunn and Charles, 1937). Lines selected for intermediate amounts of spotting are much more variable. This situation appears to reflect the fact that whereas most of the variability in the all-white and predominantly pigmented s/s stocks is due to nongenetic causes, both genetic and nongenetic factors influence the variability in the intermediately spotted stocks.
To determine whether these different lines had different piebald alleles or possessed the same "main gene," s, with different sets of "modifiers," Dunn and Charles raised an F2 population after crossing an all-white line with a dark line. 7 It follows that if distinct s alleles were responsible for the different amount of spotting in the two parental stocks, this F2 population should display a trimodal distribution and the extreme parental types should not be recovered. In actual fact, however, these types were not present as the F2, like the F1, displayed intermediate amounts of spotting with a unimodal distribution, though it was more variable than the F1. It thus appears that the all-white and mostly pigmented piebald lines did not possess different " s" genes but differed by a number of "modifying" genes which recombined freely with each other and had cumulative effects. This conclusion was substantiated further by the results of backcrossing F1 animals to the all-white line. Indeed, a rough estimate from this backcross indicated that at least three modifying genes were involved ( Grüneberg, 1952).
To study the nature of these "modifiers," Dunn and Charles outcrossed their all-white s/s mice with mice that putatively did not possess any spotting genes at all. Since the F1s from such crosses were either fully pigmented or had very small spots on the belly or forehead, it seemed that the all-white type behaved very nearly as a complete recessive when s and all the "modifying" factors were heterozygous. When these F1 animals were backcrossed to animals of the all-white stock two groups of spotted mice, corresponding to the genotypes s/+ and s/s, were evident. Although the s/s group possessed much more spotting than the s/+ group, nevertheless, animals of the latter group displayed much more dorsal white spotting than the F1, presumably because some of the "modifiers" were really not modifiers in the strict sense but were spotting genes in their own right. Dunn and Charles assigned the name of " k" genes to these modifiers and, starting from this first backcross generation, repeatedly backcrossed the lighter s/+ animals to the all-white stock. In this way a population was established in which all of the " k" genes were homozygous in both s/+ and s/s animals, a situation which was evidently complete, or nearly so, from the fourth backcross onward.
To separate s from the " k" genes, s/+ animals from the second and later backcross generations were mated inter se. Such matings produced two kinds of phenotypes: those which were all-white, and presumably s/s, constituted one-quarter of the population while the remaining, presumably s/s, constituted one-quarter of the population while the remaining, presumably s/+ (1/2) and +/+ (1/4), genotypes were dark spotted animals. By individual tests the +/+ animals were identified; they ranged from 10 to 35% dorsal white, with a mean of 18.9%. On the other hand the amount of dorsal white spotting in the s/+ animals ranged from 15 to 60% with a mean of 32.3%. Since s had been eliminated in the +/+ animals of this population their spotting must have been caused by the " k" genes introduced from the all-white line. Moreover, the fact that the s/+ animals of this same population possessed, on the average, more spotting than the +/+ mice indicated that s, in the presence of homozygous " k" genes, is not completely recessive. On the background of these genes, the three genotypes +/+, s/+, and s/s displayed about 20, 35, and 100% dorsal white and, as Grüneberg points out, "the difference between 20 and 35 on the one hand, and between 20 and 100 on the other (actually a ratio 15:80) gives a rough measure for the degree of dominance of s when acting on a background of homozygous " k" genes." 8
It seems evident that in the absence of " k" genes (or in the presence of their normal allelomorphs), s is almost completely recessive to + and in the absence of s the " k" genes too are almost completely recessive. On the other hand, these " k" genes act as semidominants in the presence of s/s. In other words "the dominance of s is influenced by the presence or absence of the ' k' genes, the dominance of the ' k' genes is influenced by the presence or absence of s" ( Grüneberg, 1952).
The "k" complex has been analyzed further by Dunn ( 1942) and it seems to consist of a large number of genes which, individually, have very small effects. At least one of these genes is dominant since when spotted mice from a " k" stock were crossed with DBAs, a strain devoid of spotting and putatively devoid of " k" genes, all F1 animals were spotted either on the tail, belly, or both. The exact number of dominant " k" genes is not known, but the frequency of spotting in F2 and backcross populations of this outcross indicated that there was more than one and probably two.
Dunn also established several sublines from the F2 generation of this outcross which he subsequently inbred. Of these, two showed a type of spotting like the original " k" stock, though of lesser degree, one showed tail spotting only, and one displayed no spotting. Nevertheless, this last stock proved to be different from the unspotted DBA parental strain in that when crossed with various spotted lines, the resulting F1 exhibited much higher degrees of spotting than when the same spotted lines were crossed to DBA mice. It therefore seems evident that the extracted "self" line differed from a real "self" line by the possession of " k" genes which by themselves could not produce spotting but which, when combined with other spotting genes, could not exert a noticeable effect. As emphasized by Grüneberg such "subthreshold" spotting genes are, in a manner of speaking, "specific modifiers" in that they are not phenotypically expressed in the absence of other genes. On the other hand, they seem to differ from "minor" spotting genes which do manifest themselves only by the fact that their influence is not as great.
When mice of a given spotted (pied) strain are compared, one finds that certain areas are always unpigmented while others are invariably pigmented. The irregularities in the pattern of the spotting are therefore confined to those regions which may or may not be white. Moreover, when the frequency of pigmentation has been determined for different regions of the coat, it is not possible to plot contour lines enclosing areas which display different frequencies of pigmentation. Such areas have been mapped out by Charles ( 1938) and the reader is referred to his paper as well as to Grüneberg's book for a detailed consideration of this aspect of piebald spotting.
| For the te allele: | ||
| te Allele (MGI) | Gene (MGI) | All Alleles (MGI) |
There are a number of so-called minor spotting genes which behave as "k" genes even though it is not known whether any of them were included in the studies of Dunn and Charles. All of these genes, with the possible exception of the one described by Pincus ( 1931), exert their influence in the absence of s/s and indeed, in most cases their effect in the presence of s/s has not been investigated. Nor have the relationships of these determinants with each other been resolved.
One of these determinants occurs in the C57BR strain where it produces a small white ventral patch varying in size from total absence to about 10% of the ventral surface. In a few mice the unpigmented area sometimes extends up onto the side of the animal, and some animals exhibit some white on the tail (with or without ventral spotting). According to Murray and G. Green ( 1933) more than 50% of C57BR mice display some degree of spotting with the frequency and the size of the belly spots being somewhat greater in males than in females. The amount of spotting among littermates also seems to correlate better with the amount of spotting displayed by their sire than by their dam. The basis for these unexpected observations is not known.
Another minor spotting gene, expressed almost exclusively on the tail, occurs in the C57BL strain ( Little, 1924, Grüneberg, 1936b). This factor has been analyzed to some extent by Grüneberg who found that it behaves as a simple recessive which segregates independently of agouti. 9
Barrows ( 1939) investigated tail spotting in mice by selecting for an increased amount of it in a genetically heterogeneous stock which did not possess s. Although his study did not include any attempts to sort out the responsible factor or factors, he believes the increased spotting he observed was due to the cumulative action of several minor genes.
There are a number of minor spotting genes which appear to affect the head. One of these has been described by Pincus ( 1931) who assumed that it exerted its effects only in the presence of piebald spotting since when combined with s/s it makes most of the head and face white. However, as Grüneberg points out, his data are not inconsistent with the possibility that the gene in question (which was assigned the symbol l) may produce some spotting, though possibly very little, in the absence of s.
A gene producing a similar pattern has been described by Fisher and Mather ( 1936; see also Grüneberg, 1952) and assigned the symbol te. Grüneberg refers to this gene as "light head" and it is conceivably the same gene as the one described above. However, in this case there is no doubt that the gene is a spotting factor in its own right as te/te mice, in the absence of other spotting genes, have a few white hairs (blaze) on their foreheads. The effect of this determinant, both in the presence and absence of s, appears to be confined to the head. Moreover, unlike the factor described by Pincus, in segregating populations there appears to be a deficiency of "light head" animals, a deficiency which Fisher and Mather ascribe to reduced viability rather than to misclassification.
Little ( 1914, 1917c) also described a recessive gene which produced a small blaze of white hairs on the forehead, and occasionally a little ventral or tail spotting, in the absence of s. Sometimes this determinant, which was found in a wild population, had no phenotypic effect at all when homozygous and Little ( 1926) demonstrated that its expression was influenced by a number of modifying genes which were widely spread in various solid colored races. The relationship of this determinant to the one described by Pincus, and to "light head," is not known and it is possible that they all are genetically related if not identical.
Another, possibly different, gene which produces a head spot (referred to as "headdot") has been described and studied by Keeler and Goodale ( Keeler, 1935; Keeler and Goodale, 1936; Goodale, 1937a, 1937b, 1942, 1943, 1948). It also behaves, in general, as a recessive though some heterozygotes display a few white hairs on the forehead, and some homozygotes fail to display any spotting at all. This spotting factor seems to differ from the others in that in animals displaying spotting of the lowest grade a white spot occurs on the forehead only, and this is the most common type. As the amount of head spotting increases, however, white appears successively on the tip of the tail, lower lip, belly, and feet. This character too is influenced by modifiers. 10
Since the pigmentation of the coat results from the synthesis and deposition of melanin by neural crest-derived pigment cells in the hair follicle, the behavior of transplants which include these components is one means of assessing their relative roles in the etiology of spotting. Accordingly Mayer ( 1965, 1967a, 1967b) investigated the basis of piebald spotting by combining very small sections of neural crest-containing neural tube with small pieces of embryonic skin of the same or different s genotype. The relative importance of these components in the development of pigment was judged by the patterns of pigmentation they produced when allowed to differentiate in the coelom of White Leghorn chick embryos. For these experiments Mayer employed a stock of a/a;s/s mice which displayed large areas of white spotting on the belly, sides and back that were predictable in location from one litter to the next ( Figure 9-3). The neural crest-containing pieces of neural tube always were taken from 9-day-old embryos at the level of the hind limb bud (or posterior to it), a region which was potentially pigmented in the s/s strain employed. The skin was derived from the ventrolateral flank region between the limb buds of embryos of varying ages, a region that was destined to be unpigmented in the piebald stock.
In his first series of experiments Mayer ( 1965) combined s/s neural tube with 11-day-old neural crest-free +/+ (C57BL/6) skin. He also combined +/+ neural tube with either s/s or with +/+ neural crest-free skin. He found that whereas grafts composed of +/+ neural tube and s/s or +/+ skin always produced pigmented hairs, as well as melanocyte populations in the dermis of the graft and in the chick tissues surrounding the graft, grafts of s/s neural tube and +/+ skin produced only pigmented hairs. Mayer also noted that some tissues, e.g., choroid, harderian gland, leg musculature, were pigmented in the +/+ but not in the s/s mice he employed (see Table 9-1). On the basis of these observations he concluded that the primary action of s is in the melanoblast but that its effect can be discerned only in certain tissues, i.e., that different tissues, or different regions of the same tissue, such as skin, vary in their capacity to promote the differentiation of s/s melanoblasts.
To substantiate this hypothesis further Mayer initiated a second series of experiments ( Mayer, 1967a) in which he combined s/s neural tube with neural crest-free s/s skin. He found that although, as in the case of s/s neural tube and +/+ skin, melanocytes never occurred in the intrafollicular dermis or in the host tissues surrounding the grafts, unlike the former combination, they also rarely occurred in hair follicles. Thus in contrast to the behavior of grafts composed of s/s neural crest and +/+ skin, composites which led to pigmented hairs in 21/31 cases, combinations of s/s neural crest and s/s skin produced pigmented hairs in only 4/27 transplants.
According to Mayer there are two possible explanations 11 for these results both of which involve the participation of the skin in the etiology of piebald spotting. One possibility is that s acts not only in the neural crest but also in the skin. The other possibility, which Mayer views as more likely, is that the neural crest is affected by the piebald gene, and that other genetic factors, e.g., members of the " k" complex, operating within the piebald stock but absent in the +/+ (C57BL/6J) strain, are responsible for the deleterious influence which the s/s skin has on melanoblast differentiation. 12
In a final series of experiments Mayer ( 1967b) combined 9-day-old s/s neural tube with potentially white areas of s/s skin of different ages and found that as the age of the skin increased so did the amount of pigment. Whereas 11-day-old s/s skin produced pigmented hairs in only 16% of the cases (and no pigment cells were observed in the intrafollicular epidermis), and 13-day-old skin produced pigmented hairs in 79% of the grafts (and again no pigment cells occurred in the epidermis), grafts recovered from implanting embryonic skin 16 days and older produced pigment regularly both in the hair follicles and in the skin. Mayer believes these experiments indicate that there is an inhibitor of melanoblast differentiation or survival in 11-day-old s/s skin and that this inhibitor gradually disappears.
Although Mayer's interpretation of his findings contrasts with Mintz's hypothesis which proposes that piebald spotting results solely from the demise and partial replacement on inviable melanoblast clones with viable cells, and does not involve any direct effect on the skin (see Chapter 7, Section VII), it should be borne in mind that his interpretation also is based upon certain assumptions which are not in accord with her hypothesis and therefore are debatable. For example, while Mayer assumes that his s/s neural tube grafts were comprised of a uniform population of (viable) melanoblasts because they were derived from a potentially pigmented area, it could be argued that cells of both viable and inviable clones of melanoblasts, and in some cases of only inviable clones, were included in these transplants (especially since the s/s mice employed were predominantly unpigmented). This possibility is based upon Mintz's contention that spotted phenotypes are a consequence of viable melanoblasts partially replacing inviable cells, a supposition which implies that there may be little or no relationship between the location of viable cells in a 9-day-old embryo and the location of pigmented areas in a mature, white spotted, mouse. 13 Thus, while Mayer's results indicate that piebald spotting cannot be attributed to a defect in the migration of s/s melanoblasts, and that many of these cells are inviable, they are not as convincing in demonstrating that the skin plays a direct role in determining their survival.
Further evidence that the primary effect of s and sl is on the neural crest stems from the megacolon condition associated with these alleles. As already noted this abnormality appears to result from a marked reduction of the myenteric plexus and this plexus is known to originate in the neural crest ( Yntema and Hammond, 1954). 14
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