Hereditary white spotting in the mouse may be caused by genes at over a dozen loci. It is thought that some genes achieve their effects by acting through the melanoblasts, and others by acting through the host tissue. The genes mi, Miwh, Wv and s are believed to belong to the former category. But it is not known how the abnormality of the melanoblasts is transformed into the spotting patterns observed. According to one view, the capacity of the melanoblasts to respond to some melanogenesis-promoting factor in the host tissues is impaired, and as all melanoblasts are affected about equally, the pattern depends on normal variations in the distribution of this factor in the host tissue. According to another, a proportion of the melanoblasts are ‘preprogrammed’ to die before differentiation. Both views are largely based on studies on the coat. It was thought that an investigation of the spotting patterns in the choroid, the Harderian gland and the inner ear might throw fresh light on the problem. This was carried out in the genotypes + /mi, Miwh+, Wv/+, Wv/Wv and s/s. The results provide strong support for the view that all melanoblasts are affected and the host tissue plays an important role in determining the pattern of spotting. However, there appear to be some indications that all melanoblasts may not be affected to the same degree.

Hereditary white spotting is common in mammals. It has been most extensively studied in the mouse, in which a large number of spotting genes at over a dozen loci are known (Searle, 1968). The effects of nearly all these genes are highly variable, depending to a great extent on the genetic background, but in many cases there is a recognizable pattern. When the genetic background is homogeneous the pattern is more regular, but some degree of variability always remains. The spots are rarely if ever symmetrical, and neither midline acts as a barrier. The situation is further complicated by the dilution or variegation of the pigmented regions in some genotypes. In extreme cases the entire animal is white. Such animals are regarded as having one very large spot and not as albinos, for white spotting and albinism are fundamentally different (Billingham & Silvers, 1960).

The genesis of white spots is generally regarded as one of the most complex problems in developmental biology. Broadly, two types of explanation have been offered: the spot could result from either some abnormality of the melano-blasts themselves, which affected their division, migration, differentiation or viability; or some abnormality of their host tissue which affected the entry, division, differentiation or viability of these cells. For long it was not possible to say which type of explanation applied to which gene, but recently considerable progress has been made in this direction, mainly as a result of the introduction of new techniques. Mayer and colleagues have concluded from their grafting experiments that the genes lethal spotting (Is;Mayer & Maltby, 1964), piebald (s;Mayer, 1965, 1967a, b) and viable dominant spotting (Wv; Mayer & Green, 1968; Mayer, 1970) act on the melanoblasts, and the genes belted (bt;Mayer & Maltby, 1964) and steel (SI;Mayer & Green, 1968; Mayer, 1970) on the host tissue. Mintz’s (1967, 1969) analysis of appropriate types of allophenic mice has led her to the view that dominant spotting (W) and the Miwh and Mibw alleles of microphthalmia (mi) act on the melanoblasts. But even where the melanoblasts have been implicated the nature of their abnormality remains obscure. Mayer (1967a, b) has suggested that the gene 5 affects the capacity of the melanoblasts to differentiate into melanocytes in response to a melano-genesis promoting factor in the host tissue, and that the pattern of spotting is determined by the normal regional variations in the concentration of this factor in the host tissue, all melanoblasts being affected equally. Mintz (1969), on the other hand, believes that in spotted genotypes a proportion of the melanoblasts are ‘preprogrammed’ for death, and does not assign any role to the host tissue.

Both these explanations are largely based on analyses of the pigmentation of the coat only. But it was recently discovered that spotting genes affected the pigmentation of the inner ear in a manner that had no clear relationship with their effects on the coat (Deol, 1970). This seemed to call for a new look at the problem based on pigmentation patterns in some other internal structures in mutants in which the melanoblasts had been implicated. The choroid was chosen because not only is it heavily pigmented in normal mice, but it permits, when spotting occurs, determination of whether there is a tendency towards a regular pattern. The Harderian gland was selected because it is one of the best organs for the study of melanocyte morphology. Both these structures had been examined before in some of the genotypes used here (Markert & Silvers, 1956), but not with regard to pigmentation patterns.

This study is confined to four genes, two of which are allelic. The following brief descriptions of their major effects apply only to our own stocks, in which the genetic background is heterogeneous and the colour background either agouti or non-agouti (for fuller accounts see Grüneberg, 1952).

Microphthalmia

The heterozygotes ( + /mi) often have completely normal pigmentation as far as external features are concerned, but spots at the end of the tail and along the mid-ventral line of the trunk are common. Mid-dorsal spots on the head also occur sometimes. The homozygotes (mi/mi) are entirely white. Their eyes are extremely reduced or absent, and the skeleton is abnormal. They die young.

Microphthalmia white

The heterozygotes (Miwh/ + ) always have a mid-ventral spot of variable size on the trunk. Scattered spots of different sizes may sometimes occur on the back in the lumbar region. The rest of the coat is appreciably diluted. In rare cases normally pigmented ‘spots’ occur (Schaible, 1969). The homozygotes (Miwh,/Miwh) are wholly white, have greatly reduced eyes, and are sterile.

Viable dominant spotting

The heterozygotes (Wv/+ ) always have a mid-ventral spot of variable size on the trunk, and quite frequently a small mid-dorsal spot on the head in addition. The rest of the coat is diluted, but in a way different from that of Miwh/ + mice. The homozygotes (Wv/Wv) are wholly white, although pigment may occasionally be found in the skin of the ear pinna, but not in the hair. They are also sterile and anaemic.

Piebald

The heterozygotes ( + /s) are as a rule normally pigmented except for the tail-tip and the digits. The homozygotes (5/5) have widespread spots, some very large, which tend to favour certain regions, and so display a moderately regular pattern. On the whole, the underside is more affected than the top.

Of the seven spotted phenotypes possible, only five were studied. Miwh/Miwh and mi/mi mice were left out because in them the structure of the eye is also abnormal. The question of normal ( + / + ) controls was not easy to decide, for it was suspected that some spotting genes may not have any external effects. In the event, control animals were taken from the inbred strains C57BL/Gr and CBA/Gr as well as from crosses between them and between them and the strain A/Gr. Altogether, the choroid was examined in 82 + / +, 24 + /mi, 14 Miwh/+, 32 Wv/+ (half with head spots and half without), 15 WV/WV and 28 s/s mice. The Harderian gland was examined in 17 + /+, 11 + /mi, 10 Miwh,/ +, 10 Wv/+, 6 Wv/Wv and 10 s/s mice. The pigmentation of the inner ear was studied only in 10 s/s mice, for the situation in the other genotypes was already known (Deol, 1970).

The eyes were marked with a fine hot needle before removing so as to establish the dorso-ventral plane. They were fixed in 10 % formol-saline because it was earlier discovered that this fixative tends to separate the retina from the choroid, which makes it unsuitable for most types of work on the eye but ideal for the present purpose. The eyes were then transferred to 70 % alcohol, and cut into dorsal and ventral halves with the aid of the cauterized spots. The retina was removed from each half, its pigmented epithelium remaining attached to the choroid. The pigmentation of the choroid could then be examined without any further treatment, there being no interference from the retinal epithelium. Its pattern was recorded on outline drawings of the type shown in Figs. 1 and 2. The Harderian gland was fixed in 10 % formol-saline, dehydrated and cleared in methyl salicylate. In addition, a few eyes and glands from each genotype were fixed in Bouin’s fluid and sectioned. The technique for the study of the pigmentation of the inner ear has already been described (Deol, 1970). Before removing the tissues the appearance of the coat was noted, and outline drawings were made in cases of heavy spotting.

Fig. 1.

Schematic drawings of the upper and lower halves of the right eyes of normal (A and B), + /mi (C), Miwh/ 4-(D), Wv/ + (E) and s/s (F) mice, showing pigmentation of the choroid, c = cornea; ch = choroid; i = iris; on = optic nerve.

Fig. 1.

Schematic drawings of the upper and lower halves of the right eyes of normal (A and B), + /mi (C), Miwh/ 4-(D), Wv/ + (E) and s/s (F) mice, showing pigmentation of the choroid, c = cornea; ch = choroid; i = iris; on = optic nerve.

Fig. 2.

Schematic drawings of the upper and lower halves of the left and right eyes of four s/s mice, showing the irregular disposition of the spots in the choroid.

Fig. 2.

Schematic drawings of the upper and lower halves of the left and right eyes of four s/s mice, showing the irregular disposition of the spots in the choroid.

The choroid

In normal mice the choroid was heavily but not uniformly pigmented, the larger blood vessels standing out fairly clearly. There were often one or more small, scattered areas where the pigment was considerably reduced or altogether missing (Fig. 1 A, B). These areas were generally not very sharply defined, and they were virtually confined to the dorsal half of the eye, although they did not favour any particular site. There was no correlation between the two eyes with regard to the size, number or disposition of these small spots.

In + /mi mice the pigment was missing from such a large part of the choroid (usually much more than half of the total area) in all cases, that the pigmented areas appeared as black spots on a clear background (Fig. 1C). They were on the whole more frequent in the vicinity of the optic nerve or along the base of the iris, but apart from this there was no discernible tendency towards a regular pattern. Although the density of the pigment in these areas appeared to be normal it may not have been so in reality, for the blood vessels stood out much more prominently. The border between pigmented and unpigmented regions was sharp but deeply indented. As in normal mice, there was more pigment in the ventral than in the dorsal half in nearly all cases, and the two sides were not symmetrical.

In Miwh/+ mice the density of the pigment was reduced throughout the choroid (Fig. 1D). The reduction, although always striking, was far from uniform, and in some regions there was no pigment at all. Patches of different intensity merged into each other gradually. Again, there was generally more pigment in the ventral than in the dorsal half, and no tendency towards a pattern or symmetry was discernible.

In Wv/+ mice the choroid often had one or more moderately large unpigmented patches of an extremely irregular shape, the total unpigmented area being always less than half (Fig. 1E). 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, and there was generally more of it in the ventral half than the dorsal. There was no tendency towards a regular pattern or symmetry. The spotting of the choroid was strikingly heavier in animals with mid-dorsal head spots than in those without them. In WV/Wv mice the choroid was totally unpigmented.

In s/s mice the degree of spotting was extremely variable, but on average the pigmented and unpigmented parts were about equal in extent (Fig. 2). In the majority of cases there was one large spot and several small ones, and in some regions there was an intermingling of minute heavily pigmented and lightly pigmented areas. The borders of the spots were usually indented, but in rare cases they ran along blood vessels and so were fairly straight (Fig. 1F). The intensity of pigmentation in most pigmented areas appeared to be normal. Again, there was more pigment in the ventral half, and no tendency towards a regular pattern or symmetry (Fig. 2).

Harderian gland

In normal mice the Harderian gland was heavily pigmented. The pigment was contained within the melanocytes, which were scattered in the connective tissue separating the lobules or in the covering sheath of the gland. The melanocytes were very large and heavily branched. In + /mi mice their number was greatly reduced, and their distribution very uneven. They were completely missing in some places, but these spots did not appear to favour any particular part. The remaining melanocytes were mostly smaller than normal and less dendritic. In Miwh/+ mice the gland was unpigmented, and not a single unmistakable melanocyte could be identified, although in sections a few granules could occasionally be seen which might have been lightly melanized melanosomes. In Wv/Wv 4-mice the number of melanocytes was only slightly reduced, and they were fairly evenly distributed. But they were on average smaller than normal, although cells of normal size were quite common. As there were only three animals with head spots in the sample, no comparison of the two types of Wv/+ mice was possible. In Wv/Wv mice the gland was wholly unpigmented. In s/s mice it seemed to be totally unpigmented in the majority of cases, and in others the number of melanocytes was severely reduced. The remaining cells were unevenly distributed, and considerably smaller and less dendritic than normal, although fairly normal cells were also observed occasionally.

The inner ear

The pigmentation of the inner ear in normal, + /mi, Miwh/+ Wv/+ and Wv/Wv mice has been described before (Deol, 1970), andmay only be summarized here. In normal mice the pigment occurred in the membranes of the inner ear in certain well-defined regions. In + /mi and Wv/ + mice it was sometimes missing from some of these regions, but not from the whole ear. In Miwh/ + mice either there was no pigment at all in the ear or it was greatly reduced in density and found only in some of the regions. In WV/WV mice about one-third of the ears had no pigment at all, and the others had it missing from some of the regions, in particular from the area just external to the lateral crista. These abnormalities of pigmentation were frequently asymmetrical.

There is a peculiar aspect of the absence of pigment from the inner ear that has not been described before. In totally unpigmented ears the appearance of the membranes that would normally be pigmented is different from that in the normal or albino ears. They are strikingly thicker, and may also have a larger number of nuclei. However, when the ear is unpigmented in certain regions only, the remainder being pigmented, then the appearance of the membranes in the unpigmented regions is not affected. This is illustrated in Fig. 3 with reference to the area around the lateral crista in Wv/Wv mice : of the 66 ears examined 45 were partly pigmented and conformed to Fig. 3B, and 21 were totally unpigmented and conformed to Fig. 3C, there being no exceptions.

Fig. 3.

Membranes of the inner ear on the medial (left) and lateral (right) sides of the lateral crista in normal (A), Wv/Wv (B and C) and albino (D) mice. Pigment (arrows) occurs on both sides in A, on the medial side only in B, and on neither side in C and D. Note that the appearance of the membranes on both sides is different in C. × 340.

Fig. 3.

Membranes of the inner ear on the medial (left) and lateral (right) sides of the lateral crista in normal (A), Wv/Wv (B and C) and albino (D) mice. Pigment (arrows) occurs on both sides in A, on the medial side only in B, and on neither side in C and D. Note that the appearance of the membranes on both sides is different in C. × 340.

In s/s mice, of the 20 ears examined one was found to be 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.

The effects of the genes mi, Miwh, Wv and s on the coat, the choroid, the Harderian gland and the inner ear are summarized in Table 1. It is clear that external pigmentation is no reliable guide to internal pigmentation. Moreover, no general trend emerges when the effects of different genes are compared. It is highly improbable that each structure is colonized by its own ‘species’ of melanoblast, and explanations based on anomalous migration of melanoblasts appear even more unsatisfactory now than they did when they were carefully considered by Markert & Silvers (1956) and rejected. The spotting patterns of the inner ear clearly favour Mayer’s (1967a, b) view that all melanoblasts are about equally affected and the spotting pattern depends on the normal regional variations in the distribution of some melanogenesis-promoting factor in the host tissue, and are difficult to reconcile with Mintz’s (1969) view that a proportion of the melanoblasts are ‘preprogrammed’ for death. For instance, in all of the 45 pigmented ears of the Wv/Wv genotype that were examined pigment was missing from the lateral side of the lateral crista but was present on the medial side (Fig. 3B). The region affected is so minute that the number of melanoblasts involved must be very small. In order to fit this observation into Mintz’s scheme it would have to be assumed that melanoblasts for the colonization of this minute region are always precisely earmarked beforehand, so that only the doomed ones get to it. This appears to be unlikely. In all probability, melano-blasts from any particular part of the neural crest go only approximately to the same site, not exactly. Mayer’s hypothesis requires the simpler assumption that the two sides of the crista differ with regard to the melanogenesis-promoting factor, so that the same abnormal melanoblasts that cannot differentiate on the lateral side can do so on the medial side. The importance of the host tissue is also indicated by the strong tendency of the choroidal spots to favour the dorsal half, observed in normal as well as mutant mice.

Table 1.

Summary of the effects of the genes mi, Miwh, Wvand s on the pigmentation of the coat, the choroid, the Harderian gland and the inner ear (inconstant features are given in parentheses)

Summary of the effects of the genes mi, Miwh, Wvand s on the pigmentation of the coat, the choroid, the Harderian gland and the inner ear (inconstant features are given in parentheses)
Summary of the effects of the genes mi, Miwh, Wvand s on the pigmentation of the coat, the choroid, the Harderian gland and the inner ear (inconstant features are given in parentheses)

The role of the host tissue implies that the spots will form a regular pattern, for the distribution pattern of the melanogenic factor, being a normal feature of the tissue concerned, must be regular. But the disposition of the spots in the choroid, apart from the tendency mentioned above, was quite irregular in all mutants. Nor was there any sign of a regular pattern in the Harderian gland. This might mean that although all melanoblasts are affected they are not affected to the same extent, so that some of them can differentiate where others cannot, a point implicit in Mintz’s (1969) view. If so, one would be forced to the conclusion that the complete pigmentation pattern of a mutant is the product of non-uniform effects of the gene on the melanoblasts and the normal variations in the distribution of the melanogenesis-promoting factor, which would be of little scientific value, however close to the truth it might be.

Mayer’s assumption of a melano-genesis-promoting factor with variable distribution is well founded, for in normal mice pigment occurs only in certain organs or certain regions of certain organs, sometimes sharply defined. In view of the remarkable migratory powers of the melanoblasts it is unlikely that other parts remain unpigmented because these cells cannot reach them. Moreover, melanocytes in different tissues often differ in size and other morphological features. But the results of this study suggest that the melanogenic factor may not be a simple entity. If it were, then certain general trends would be detectable when the effects of different genes are compared, which is not the case. It would seem that a complex of several factors is involved, and that different tissues and even different parts of the same tissue may vary with regard to not only the concentration of the complex but also its composition, each spotting gene impairing the capacity of the melanoblast to respond to some particular component or components of the complex.

It is generally assumed that melanoblasts that fail to differentiate die. This is because in the hair follicles in unpigmented regions no cells can be identified that may be regarded as abnormal melanoblasts or amelanotic melanocytes. There is no reason to challenge this view, but the fact that the appearance of the ‘spotted’ membranes is clearly and consistently different in totally unpigmented and partially pigmented ears, but similar in partially pigmented and albino ears (Fig. 3), argues that the situation may not always be so simple. Moreover, some tissues in + mice may have only very lightly pigmented melanocytes or just a few pigment granules here and there. This shows that melanoblasts that have barely crossed the threshold of differentiation can survive at least in some cases, and suggests that melanoblasts that have just failed to do so may also not always die. Further work on this mutant may prove rewarding.

As the host tissue plays an essential role, some genes may cause spotting by affecting this factor. Indeed, it has been suggested that the genes belted (bt;Mayer & Maltby, 1964) and steel (SI;Mayer & Green, 1968; Mayer, 1970) act in this manner. The evidence for SI appears to be strong, but the case for bt is rather weak, for it is based on the observation that in bt/bt mice in spotted regions melanocytes occur in the dermis but not in the follicles, which can be equally well explained on the basis of melanoblast impairment. It may also be mentioned that certain types of hyperpigmentation, such as that observed in the PET/MCV strain in which pigment was found in all parts of the body with the exception of the gut mucosa (Nichols & Reams, 1960), could be profitably studied if viewed as spotting in reverse.

The melanocytes in the anterior layer of the iris are of choroidal origin. Heterochromia of the iris occurs when the choroidal spots extend into the iris, and its anterior layer becomes unpigmented partially or asymmetrically. The condition is sometimes observed in the absence of any spotting of the skin or hair, as in Waardenburg’s syndrome in man (Waardenburg, 1951). In the light of the present study it is clear that it points to the presence of a spotting gene, which in the circumstances has affected only internal pigmentation. Very light eyes in dark-haired persons may also be sometimes due to the same cause, the iris being wholly affected on both sides in this case.

It has recently been suggested that the melanocytes of the inner ear may be of the same origin as those of the retina (Theriault & Hurley, 1970), and so essentially different from those of other organs. This is based on the observation that the shape of the melanosomes (pigment granules) is similar in both. This suggestion ignores the fact that the melanocytes of the retina are unusual in their forming an epithelium, being non-secretory, having no dendrites and being unaffected by spotting genes as such. The melanocytes of the inner ear, on the other hand, resemble those of other organs in that they do not form an epithelium, are secretory, have dendrites and are affected by spotting genes. It is much more likely that the melanocytes of the retina and the inner ear have similar melanosomes because they are active at about the same time in development and long before others, as the same authors have also found.

The author is grateful to Dr Gillian M. Truslove for her help in the collection of the material, and to Mrs Patricia Beveridge for technical assistance. His thanks are due to Mr A. J. Lee for the drawings.

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