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Transcript
/ . Embryo], exp. Morph. Vol. 26, l,pp. 123-133, 1971
123
Printed in Great Britain
Spotting genes and internal pigmentation
patterns in the mouse
By M. S. DEOL 1
From the Department of Animal Genetics, University College London
SUMMARY
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 +1 mi, Miwh/ +, Wv/ +, Wvj 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.
INTRODUCTION
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 melano1
Author's address: Department of Animal Genetics, Wolfson House, 4 Stephenson Way,
London, NW1 2HE, U.K.
124
M. S. DEOL
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, 1961a, 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 (1967 a, b) has suggested that the gene s affects the capacity of
the melanoblasts to differentiate into melanocytes in response to a melanogenesis 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.
MATERIAL AND METHODS
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 Griineberg, 1952).
Micr ophthalmia. 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
Internal pigmentation patterns
125
spots on the head also occur sometimes. The homozygotes (mijmi) are entirely
white. Their eyes are extremely reduced or absent, and the skeleton is abnormal.
They die young.
Microphthalmia white. The heterozygotes (Miwhj +) 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 (MiwhjMiwh) are wholly white, have greatly reduced eyes, and are
sterile.
Viable dominant spotting. The heterozygotes (W°\ +) 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 Miwhj + mice. The homozygotes (WvjWv) 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 (s/s) 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 Miwhl +,
32 Wvl+ (half with head spots and half without), 15 Wv/Wv and 28 4y mice.
The Harderian gland was examined in 17 + / + , 11 + jmi, 10 Miwhj +, 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
126
M. S. DEOL
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.
RESULTS
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 +1 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 Miwhl+ 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 Wvl+ 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 sjs 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
Internal pigmentation patterns
Left eye
Fig. 1
127
Right eye
Fig. 2
Fig. 1. Schematic drawings of the upper and lower halves of the right eyes of normal
(AandB), +1 mi (C), Mi11*/ + (D), Wvl+ (E) and s/s (F) mice, showing pigmentation
of the choroid. c = cornea; ch = choroid; / = 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.
128
M. S. DEOL
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 + jmi 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 Miwhl+ 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\ + 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
Wvl+ mice was possible. In WvjWv mice the gland was wholly unpigmented.
In sis 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, + Imi, Miwhl + , Wvj + and
Wvl Wv mice has been described before (Deol, 1970), and may only be summarized
here. In normal mice the pigment occurred in the membranes of the inner ear in
certain well-defined regions. In + Imi and Wvl + mice it was sometimes missing
from some of these regions, but not from the whole ear. In Miwhl + 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 WVIWV 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
Internal pigmentation patterns
129
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. x 340.
EMB 26
130
M. S. DEOL
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.
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.
Table 1. Summary of the effects of the genes mi, Mi wh , W v and s on the pigmentation of the coat, the choroid, the Harderian gland and the inner ear {inconstant
features are given in parentheses)
Genotype
Coat
Choroid
Harderian
gland
Inner ear
+ lmi
Severely spotted
Severely spotted
(Lightly spotted)
Severely, unevenly diluted
Unpigmented
Moderately
spotted
Fairly normal
Unpigmented or
diluted or
spotted
(Lightly spotted)
W°\WV
(Spot on head,
belly, tail)
Spot on belly.
Diluted. (Spots
on back)
Spot on belly.
Diluted. (Spot
on head)
Unpigmented
Unpigmented
Unpigmented
s\s
Widespread spots Severely spotted
Miwhl +
wvi+
Unpigmented or
severely spotted
Unpigmented or (Moderately
severely spotted spotted)
DISCUSSION
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
Internal pigmentation patterns
131
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, melanoblasts 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.
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 melanogenesis-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
9-2
132
M. S. DEOL
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 Miwhl + 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.
Internal pigmentation patterns
133
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{Manuscript received 5 February 1971)