Chapter 12

Microphthalmia and Other Considerations

I. Microphthalmia Locus

Besides dominant spotting ( W) and steel ( Sl) there is another series of alleles which frequently produce white spotting when heterozygous, and usually completely eliminate pigment from the coat, and sometimes from the eyes as well, when homozygous. These alleles occur at the so-called "microphthalmia locus" (chromosome 6) and include the following.

A. Microphthalmia ( mi)

This allele was recognized first in the grandchildren of a male which had received 1500 r of X-irradiation (Hertwig, 1942a, 1942b). Because it is not known whether the son of this male carried the gene, or whether it was introduced by the female to whom he was mated, it is uncertain whether the mutation was induced or occurred spontaneously. Its effects have been described in some detail by Grüneberg ( 1952) and much of the following is based on his account. mi, although not written with a capitol first letter, is a semidominant gene which is usually lethal when homozygous.

1. Effect When Heterozygous

When heterozygous with the wild type allele mi has no noticeable effect on the intensity of the coat ¹ but does influence the pigmentation of the eye; mi/+ newborns display less iris pigment than +/+. ² These heterozygotes also may display some white spotting ( Grüneberg, 1948), a manifestation which seems to depend upon their genetic background. Grüneberg found that when a mi/+ male in which spotting was not recognized was outcrossed to two +/+ females from a stock which carried no major spotting genes, ³ that most of their mi/+ but none of their +/+ offspring exhibited unpigmented tail tips of varying length. Additional spotting on the head and/or belly and chest occurred in mi/+ mice of the F₂ and subsequent generations, while some +/+ animals in these populations also displayed some low grade tail spotting. The amount of spotting on the head of the mi/+ mice was quite variable. It always occurred near the midline of the head and varied from a few white hairs on the forehead to fairly extensive white wedges which included a large part of the whisker area. The amount of belly spotting also varied. In some instances it was characterized by long and narrow, nearly symmetrical, streaks along the midline of the belly or chest, while in others it covered up to one-quarter of the ventrum. No dorsal spotting was ever observed. An analysis of the frequency of the spotting in these mi/+ mice indicated that at least three genes were involved. Some of these were minor spotting genes (see Chapter 9, Section II, F), which are very common in mice (Grüneberg, 1936b, 1948), whose effects were greatly intensified in mi/+ animals.

While mi/+ mice vary as to whether they display any external spotting, and as to the degree of this spotting, the internal pigmentation patterns of these heterozygotes seem to be more predictable. Thus Deol ( 1971) observed that pigment was absent from such a large part of their choroid (usually significantly more than half of the total area) that the pigmented areas appeared as black spots on a clear background. These were, in general, more frequent in the area of the optic nerve or along the base of the iris, but aside from this there was no obvious tendency toward a regular pattern. Deol also reported that the number of melanocytes in the harderian gland of these heterozygotes was greatly reduced and their distribution was very uneven. They were missing completely in some places, but not in any particular area. The melanocytes themselves were mostly smaller and less dendritic than in +/+ mice. Although Deol ( 1970b) did not find any abnormalities in the inner ear of these animals, about a quarter of the ears examined had no pigment in the lateral crista. The effects of mi/+ on pigmentation are summarized in Tables 12-1 and 12-2.

2. Effect When Homozygous

Homozygous mi/mi mice are completely devoid of pigment both in the fur and in the eyes and in this respect they mimic albino animals. However, unlike the albino, the unpigmented condition of these animals is undoubtedly the consequence of an absence of melanocytes. The eyes of these homozygotes are reduced in size and usually covered with cataracts ( Tost, 1958); the eyelids never open and there is a coloboma of the retina ( Muller, 1950). These anomalies result from the fact that during development the optic vesicle fails to form a proper cup and the choroid fissure remains permanently open (Muller, 1950, 1951; see also Fischer and Tost, 1959; Konyukhov and Sazhina, 1963, 1966; Konyukhov et al., 1965). The inner ear of these homozygotes also displays abnormalities; ⁴ it also possess no pigment ( Deol, 1970b).

The most severe consequence of mi when homozygous is that, like grey-lethal ( gl) (see Chapter 5, Section I, B), it results in an impairment of secondary bone resorption. This osteopetrosis, although not as severe as in grey-lethal, nevertheless usually is lethal. Thus in the large majority of mi homozygotes the incisors never erupt and as a consequence they die on weaning. Occasionally one or two incisors are cut and if they enable the animal to eat solid food the mouse may live for some time. A microphthalmic male of Hertwig's lived to breed. Another influence of mi/mi mentioned by Hertwig is that it causes a slight curliness of the vibrissae.

The reshaping of the bones in normal mice and the influence of mi (and gl) on the process has been studied in some detail by N. Bateman ( 1954). Grüneberg ( 1948) also investigated the skeleton and teeth of mi homozygotes and found that these animals were "an extraordinarily faithful mimic of gl." In both homozygotes secondary bone absorption is disturbed profoundly and the teeth of mi/mi mice are almost exactly like those of the grey-lethal animal. Nevertheless, the dentition of mi homozygotes is less extremely abnormal and the failure of secondary bone absorption is less completely disturbed by mi than by gl. The differences are, however, slight.

Although the precise etiology of the osteopetrotic syndrome in microphthalmic mice is not known, there are no reasons to believe that it is different from the anomaly in grey-lethal animals. Consequently the reader is referred to Chapter 5, Section C for a more detailed treatment of the condition. ⁵

B. White ( Mi^wh)

Mi^wh was described originally by Grobman and Charles ( 1947) after it was found in an F₁ female of a C57BL female x DBA male mating. ⁶ The male had received a total of 221 r of X-rays and hence it is conceivable that the mutation was induced and not spontaneous ( Grüneberg, 1952).

1. Effect When Heterozygous with Wild Type

When heterozygous with the wild type allele Mi^wh produces a coat color very similar to dilute ( d/d) though it is slightly lighter ( Plate 3-H). The eye color of Mi^wh/+ mice is a very dark ruby on a black ( B/—) background and red in B/—;d/d animals ( Grobham and Charles, 1947). The coat color of a/a;B/B;d/d;Mi^wh/+ has been described as "pastel-grey" and of a/a;b/b;d/d;Mi^wh/+ as a very delicate silver ( Grobman and Charles, 1947).

Mi^wh/+ heterozygotes usually display spotting on their feet, the tip of the tail, and the belly. Small irregular white spots, sometimes consisting of only a few hairs, often occur also on the back in the lumbar region and very occasionally normally pigmented "spots" are found ( Schaible, 1969; see Chapter 10, note 28). In a Mi^wh stock derived from an outcross to C57BL, nearly all Mi^wh/+ mice had fairly large belly spots which were often broad and sometimes formed rudimentary belts ( Grüneberg, 1953) (see Plate 3-H). Unlike the situation in mi/+ animals, head dots or blazes do not occur. Nevertheless, the total amount of white is much greater in Mi^wh/+ than in mi/+. Grüneberg also found that on similar genetic backgrounds, Mi^wh/+ and mi/+ mice differed greatly in the incidence of ventral spots which occurred in most of the former (37 of 41) but in few of the latter (12 of 56). The shape of the belly spots was about the same in both genotypes but those of Mi^wh/+ were on the average larger. Although in the particular populations studied spotting on the head or face was not present in either homozygote, tail spotting was present in both with more of the tail tending to by unpigmented in Mi^wh/+.

Hairs from the pigmented regions of Mi^wh/+ mice exhibit what E.S. Russell ( 1946) has termed "pigmentation lag," i.e., there is a delay in the deposition of medullary pigment, especially distally, and the medullary granules vary greatly in size and in shape ( Wolfe and Coleman, 1964). According to Grobman and Charles MI^wh/+ reduces or restricts medullary pigment in a more regular manner than does dilute ( d/d). They also reported that Mi^wh/+ animals darken slightly with age, the rate of darkening being somewhat less than in d/d mice. ⁷

The inner ear of Mi^wh/+ mice either has no pigment at all or the amount of pigment is greatly reduced in density and found only in certain areas. ⁸ The inner ear itself displays severe abnormalities with every ear affected. As in the case of mi/mi no part of the cochlea duct is normal. Indeed, according to Deol ( 1970b) Mi^wh/+ animals exhibit the same ear duct abnormalities as mi/mi mice (see note 4). ⁹

Deol also found that the density of the pigment was reduced throughout the choroid of Mi^wh/+ genotypes. ¹⁰ This reduction, although far from uniform, was quite striking. In fact, some regions were pigment-free. In general, there was more pigment in the ventral than in the dorsal half of the eye ( Deol, 1971).

The harderian gland of Mi^wh/+ mice is unpigmented ¹¹ and the number of melanocytes in the nictitans is somewhat reduced ( Markert and Silvers, 1956). The effects of Mi^wh/+ on the pigmentation of the coat, choroid, harderian gland, and inner ear are summarized in Table 12-1. Its effects on the pigmentation of the eye are presented in Table 12-2. ¹²

2. Effect When Homozygous

Like mi/mi homozygotes, Mi^wh/Mi^wh genotypes are completely white and because their eyes appear pinkish they too have been described as "mock-albinos." However, as in the case of mi/mi, it must be emphasized that the etiology of this unpigmented condition stems from the absence of melanocytes ( Silvers, 1956) and not from a biochemical block in melanin synthesis (see Figure 3-11a). ¹³ These homozygotes also differ from albinos ( c/c) in that the pigmentary layer of the retina is mostly missing (where present it is somewhat reduced, they usually are smaller than albinos, and a little pigment sometimes occurs in the inner layer of the iris ( Grüneberg, 1953; Deol, 1970b, 1973). ¹⁴ Unlike mi homozygotes the skeleton of these mice seems to be quite normal ( Grüneberg, 1952). The inner ear of Mi^wh/Mi^wh displays no pigment and exhibits the same abnormalities as Mi^wh/+ (and mi/mi) genotypes ( Deol, 1970b). ¹⁵

3. Etiology of Spotting

We already have presented the evidence of Mintz (see Chapter 7, Section VII) which indicates that the complete absence of melanocytes from the coat of Mi^wh/Mi^wh mice, and their partial absence from the coat of Mi^wh/+ animals, is due to inviable clones of melanoblasts preprogrammed to die as a consequence of Mi^wh acting within these cells and not via the skin. ¹⁶ Further evidence that Mi^wh acts autonomously within the melanoblast is derived from the observation that +/+ host melanoblasts which invade transplants of Mi^wh/Mi^wh neonatal skin function normally and produce intensely pigmented hairs ( Silvers and E. Russell, 1955).

4. Effect when Heterozygous with mi

Mi^wh/mi heterozygotes also lack pigment in the coat but appear to resemble Mi^wh homozygotes more than mi/mi mice. The eyes of these animals have a pigment ring in the iris, sometimes only slight in extent and easily overlooked, but frequently sufficiently heavy to give the eye a red rather than a pink color ( Grüneberg, 1953). ¹⁷ According to Deol ( 1973) both the choroid and the retinal epithelium of these heterozygotes are unpigmented but an occasional retinal cell does possess some melanin granules. These, however, are usually misshapen and unevenly distributed. ¹⁸ The effects of Mi^wh/mi on the pigmentation of the eye are summarized in Table 12-2.

There is no evidence of any skeletal defects in Mi^wh/mi mice nor are there teeth affected. They also appear less microphthalmic than Mi^wh and, of course, mi homozygotes (Grüneberg, 1952, 1953). It thus appears that, in general, Mi^wh is completely dominant over mi (but see also note 25). Nevertheless, taken together, the relationship between these alleles is somewhat paradoxical. Thus, as pointed out by Grüneberg ( 1953), the homozygous manifestation of mi is much more extreme than Mi^wh, even though when heterozygous (with +) Mi^wh has a much greater effect on the color of the coat and on spotting than does mi. ¹⁹

C. Black-Eyed White ( mi^bw)

This allele arose spontaneously in the C3H strain ( Kreitner, 1957). It was discovered first in 1954 and was known as bw until its allelism with Mi^wh was demonstrated by Hollander in 1961. mi^bw is completely recessive to wild type. When homozygous it usually eliminates all the pigment in the coat but not the eyes, which remain black. Very occasionally a small pigmented spot(s) ("ticking") may be present on the dorsum or on the head. These may be composed of only a few hairs or comprise a pigmented area about 2 cm in diameter ( Kreitner, 1957; Hollander, personal communication). These spots have been described as black with a grayish tone "produced by variegation, and not by incomplete pigmentation of the individual hairs" ( Kreitner, 1957). According to Schaible ( 1963a, 1963b) Mi^wh/mi^wh heterozygotes are white with some pale yellow spots, of the same shade on a^e, a, + and A^y backgrounds, which fade in the adult. This compound is similar to that of MI^wh/mi^sp (see below) except that the pigmented spots apparently are more dilute and more restricted in size. It should be stressed that since Mi^wh homozygotes never display any pigment in the coat and mi^bw/mi^bw mice only rarely display small pigmented spots, it is surprising that Mi^wh/mi^bw heterozygotes show any spots at all. These heterozygotes have normal eyes except for the red pupil which is characteristic of Mi^wh heterozygotes. Very rarely (one of 136 heterozygotes examined) microphthalmia occurs ( Schaible, 1963b).

mi^bw when homozygous is evidently also a dominance modifier of P. Thus whereas mi^bw/mi^bw;P/P mice have black eyes and mi^bw/mi^bw;p/p animals are phenotypically indistinguishable from albinos, mi^bw/mi^bw;P/p genotypes are "ruby-eyed" ( Kreitner, 1957). mi^bw/mi^bw mice have normal viability, size prolificness, and litter size.

1. Etiology of Spotting

Like Mi^wh/Mi^wh <--> +/+ allophenics, mi^bw/mi^bw <--> +/+ allophenics are either fully pigmented or display white spotting. Thus Mintz( 1971a) found that, in the presence of skin mosaicism, 50% of such allophenics showed both black and white coat areas. These results indicate that, like Mi^wh, the white coat of mi^bw/mi^bw mice is due to the preprogrammed death of melanoblast clones (see Chapter 7, Section VII). Further evidence that this is the case stems from some experiments of Markert ( 1960). He implanted tissues (which normally contain melanoblasts) from either +/+ or mi^bw/mi^bw embryos into the anterior chamber of the eye of albino hosts. Whereas such implants always gave rise to many melanocytes which migrated over the inner surface of the host eye when they were derived from +/+ embryos, in no instance did melanocytes occur in or near the mi^bw/mi^bw implants. It thus appears that for some reason the neural crest of mi^bw/mi^bw mice does not possess cells capable of forming pigment, even in a favorable milieu.

D. Microphthalmia-Spotted ( mi^sp)

The behavior of this allele was described briefly by Wolfe ( 1962) and subsequently in much more detail by Wolfe and Coleman ( 1964); the following is based on their observations. mi^sp is one of the most interesting of the mi-series because it does not express itself either when heterozygous with wild-type or when homozygous, both mi^sp/+ and mi^sp/mi^sp genotypes appearing indistinguishable from +/+. However, when heterozygous with either Mi^wh or mi, new phenotypes are produced. ²⁰ On an a/a background, Mi^wh/mi^sp mice are pale yellow with white spots on both their dorsum and ventrum, and with pigmented eyes. The yellow areas become "sooty" at the first moult. mi/mi^sp mice are nearly all-white except for a variable number of faint irregular patches of pigment usually expressed as "flecks" of pigmented hairs on the dorsum. ²¹ The pigmentation of their eyes is less than that of mi/+ and nearly indistinguishable from that of C57BL/6J- ru/ru (ruby-eye) mice. The mutant was expressed first when a cross between a C57BL/6J female and a C57BL/6J- Mi^wh/+ male produced five deviants.

1. Tyrosinase Activity

Although mi^sp/+ and mi^sp homozygotes are phenotypically indistinguishable from +/+ mice, tyrosinase activity in skin slices from 5-day-old animals of these genotypes (all on C57BL/6 backgrounds) are different. Thus, as shown in Figure 12-1, +/+ displays the most activity, followed in turn by mi^sp/+, mi/+, and mi^sp/mi^sp. mi^sp/+ mice differ significantly from mi^sp/mi^sp and both differ significantly from +/+. On the other hand, mi/+ does not differ from either mi^sp/+ or mi^sp/mi^sp, but does differ from Mi^wh/+ and, as already noted, +/+. ²²

2. Hair Granules

In spite of the fact that mi^sp/mi^sp and mi^sp/+ genotypes have a lower tyrosinase level than +/+ no differences were observed in the number, kind, or arrangement of cortical granules in these genotypes, or in mi/+ mice. Nor were any differences observed in the distance to the first cortical pigment measured from the tips of the hairs. Because medullary granules were compactly grouped and could not be counted without a large error they were not compared. However, the granules in all of these deviants did not appear to differ from +/+ (in this case nonagouti, black) which are long oval and intense black ( E. Russell, 1946). ²³

Mi^wh/Mi^sp hairs displayed a pigmentation lag even more pronounced than Mi^wh/+ and in some hairs no cortical granules occurred at all. Thus most of the pigmentation of the coat of these mice stems from granules located in the proximal portion of the shaft. The medullary granules vary greatly in shape and size (from 0.4 microns to as much as 5 microns in greatest diameter, presumably because of clumping). The granules seem less dense than +/+ and have a yellowish appearance when observed by transmitted white light. ²⁴ The sizes and shapes of granules do not vary as much in postjuvenile hairs and there are more of them in both the cortex and the medulla, an observation in accord with the observations of Grobman and Charles for hairs of Mi^wh/+ mice.

E. White Spot ( mi^ws)

mi^ws, a semidominant, occurred in a C57BL/6 strain which had, over a 5-year period, been exposed to chronic gamma-irradiation at a dosage of 0.2 mrad/hr ( D.S. Miller, 1963). When heterozygous with the wild-type allele it frequently, though not invariably, produces a white spot (often in the form of a diamond) on the belly. The toes and tail tip also are commonly white and adults may become grizzled, but not diluted (Hollander, personal communication). When homozygous mi^ws eliminates all pigment from the coat, the eyes are pink (a "mock albino"). Homozygotes are viable and fertile with little, if any, microphthalmia ( Hollander, 1964; personal communication). The mi^bw/mi^ws heterozygote is a black-eyed white but the Mi^wh/mi^ws genotype (unlike Mi^wh or mi^ws homozygotes which are completely white) is obviously spotted, often checkerboard-like, with yellowish-brown (fawn) to grey. These spots gradually molt lighter so that older mice look practically black-eyed white (Hollander, personal communication). Moreover, also unlike either Mi^wh or mi^ws homozygotes (whose eyes appear pink), Mi^wh/mi^ws heterozygotes have dark eyes of normal size. ²⁵ Indeed, as pointed out by Hollander ( 1968) their phenotype is about what one might expect from the interaction of nonalleles. Nevertheless, no crossing over has been detected. These observations, therefore, give further impetus to the possibility already discussed (see notes 16 and 20) that mi is a complex locus, somewhat analogous to the brachyury ( T) series of alleles.

F. Other Alleles

In addition to the 5 mi-alleles noted above there are five others: three which have been only briefly described, red-eyed white ( mi^rw), microphthalmia-Oak Ridge ( Mi^or), and microphthalmia-brownish ( Mi^b), and two, mi^x and eyeless white ( mi^ew), which have been assigned provisional symbols.

mi^rw is a recessive allele. It occurred in the CBA/J strain ( Southard, 1974). "The original deviant was white with a pigmented ring around the neck and small red eyes." Mi^wh/mi^rw heterozygotes are predominantly white with tan spots and red eyes; mi^sp/mi^rw genotypes are wild-type but with a white belly spot; and mi/mi^rw animals are completely unpigmented either with no eyes or with very small eyes.

The allele Mi^or has been described by Stelzner ( 1964, 1966). It was noticed first in an offspring of a male whose spermatogonia had received gamma-irradiation. Mi^or/+ heterozygotes have a slightly diluted coat and pale, pinkish ears and tail. A white belly streak and/or white head spot also is frequently present. The eyes of these heterozygotes display reduced pigmentation at birth (resembling b/b) but appear full-colored in adults. Their fertility appears normal. Mi^or homozygotes have white fur. The eyes are either absent or they are so reduced in size that they appear absent. The eyelids never open and the incisors often fail to erupt or are poorly formed. Like mi/mi, animals with this latter condition usually die after weaning unless given powdered food. Males are fully fertile, but all six females tested proved sterile. This mutant seems to resemble mi in some ways and Mi^wh in others. Thus Mi^or/+ heterozygotes, like Mi^wh/+ animals, and unlike mi/+ mice, have a dilute pigmentation, reduced eye pigment and (usually) white spotting. On the other hand, the effect which Mi^or has when homozygous much more closely resembles the situation in mi/mi than in Mi^wh/Mi^wh mice. Mi^or/mi heterozygotes resemble Mi^or/Mi^or; they are white, appear to be eyeless, and so far, all have been toothless. Males are fertile.

The allele Mi^b also occurred at Oak Ridge ( Larsen, 1966), this one spontaneously in an offspring of a (101 x C3H)F₁ male x T (multiple recessive) female. The coat of Mi^b/+ heterozygotes is diluted, displaying a brownish cast; their tail and ears also are paler than +/+. Homozygotes are white with reduced eye pigment; they do not appear to be microphthalmic. The teeth are normal and both sexes are fertile. Mi^wh/Mi^b;A/A mice are light cream with white spots and ruby eyes; Mi^or/Mi^b animals are indistinguishable from Mi^b/Mi^b.

mi^x, a recessive, occurred in the NZB/Mac strain ( Munford, 1965). Animals homozygous for this mutation are black-eyed whites. Mi^wh/mi^x heterozygotes are also black-eyed whites; reduced eye size and eyelid closure of variable extent also have been observed in these heterozygotes.

mi^ew, another recessive, occurred in the C57BL/6Bn stock at Lawrence, Kansas ( Miner, 1968). mi^ew/+ heterozygotes are normally pigmented with black eyes. mi^ew homozygotes are white with undeveloped eyes (histological studies show an almost complete lack of eye development in adults) and eyelids which never open; their teeth appear normal and their fertility is somewhat reduced. Mi^wh/mi^ew heterozygotes are white with slightly reduced dark red eyes and with eyelids which are slightly closed; mi^bw/mi^ew animals are black-eyed whites; and mi^sp/mi^ew mice are white with black (on a nonagouti background) spots and normal black eyes ( Wood and Miner, 1969; H.G. Wolfe, personal communication). ²⁶

The effects of the various mi-locus genotypes considered above are summarized in Table 12-3.