/ . Embryol. exp. Morph. Vol. 30, 2, pp. 483-489,1973
Printed in Great Britain
4g3
The role of the tissue environment in the
expression of spotting genes in the mouse
By M. S. DEOL 1
From the Department of Animal Genetics, University College London
SUMMARY
Genes causing white spotting in the mouse act in two major ways: some affect the melanoblasts, while others affect the tissue environment of the melanoblasts. The question is whether
the normal tissue environment plays any role in the origin of spots in those mutants in which
the melanoblasts are believed to be the site of gene activity. An earlier study, using the
genotypes +/mi, Mi'ck/+, s/s, W/+ and WVIW° (mi = microphthalmia; Miv'h = white;
s = piebald; W = viable dominant spotting), indicated that it probably does, the evidence
largely consisting in the occurrence of extremely precise pigmentation patterns on a minute
scale. It seemed that more direct evidence could be obtained by comparing the pigmentation
of the iris with that of the choroid and the retina in the same eye in these and other genotypes.
The outer and inner layers of the iris derive their pigment cells from the choroid and the
retina respectively; therefore any clear and consistent differences between the behaviour of
these cells in their original and their secondary place of activity would constitute evidence
for the role of the tissue environment. Such differences were found.
It was also found that in another genotype, Miuh/mi, the retinal pigment cells, although
unpigmented, are clearly distinguishable. This casts serious doubt on the widespread assumption that melanoblasts which do not differentiate always die.
INTRODUCTION
Genes causing white spotting are common in mammals. In the mouse alone
over a dozen such loci, some with several alleles, are known (Searle, 1968).
Although the origin of white spots has been the subject of numerous studies, it
remains largely obscure and surrounded by controversy. There appears to be
general agreement on one point only: no melanocytes can be found in spotted
regions (Silvers, 1956; Billingham & Silvers, 1960). Whether they do not occur
at all, or are so modified as to be unrecognizable, is not known.
At one time, explanations based on faulty migration of melanoblasts were in
favour, but they were shown to be unsatisfactory by Markert & Silvers (1956).
Recent experimental studies suggest that some spotting genes express themselves
by affecting the melanoblasts in such a way that they cannot survive, proliferate
or differentiate in the tissues they normally colonize (Mayer & Maltby, 1964;
Mayer, 1965, 1967a, b, 1970; Mayer & Green, 1968; Mintz, 1967, 1971), while
others do so by affecting the host tissue so that it inhibits the entry, survival,
1
Author's address: Department of Animal Genetics, Wolfson House, 4 Stephenson Way,
London NW1 2HE, U.K.
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M. S. DEOL
proliferation or differentiation of the melanoblasts (Mayer & Maltby, 1964;
Mayer & Green, 1968; Mayer, 1970, 1973). However, it is not clear how, in
mutants in which the melanoblasts have been implicated, the spotting patterns
characteristic of the genes are produced. Mayer (1967a, b) has suggested, with
reference to the gene piebald (s), that tissues normally colonized by melanoblasts contain some melanogenesis promoting factor with regional variations
in concentration, and the gene alters the capacity of the melanoblasts to
differentiate into melanocytes in response to this factor, with the result that
although all melanoblasts may be affected to about the same extent, they would
differentiate in some regions and not in others. Mintz (1967, 1971), on the other
hand, holds the view that in spotted genotypes a proportion of the melanoblasts are 'preprogrammed' to die before differentiation, which would lead to
the characteristic pattern without any intervention by the host tissue. In an
attempt to discriminate between these viewpoints the author analysed the
pigmentation patterns in some internal organs in a number of genotypes, and
found evidence pointing to the role of the host tissue (Deol, 1971). But this
evidence was all indirect, largely consisting in the occurrence of extremely
precise pigmentation patterns on a minute scale.
It appeared that more direct evidence could be obtained by comparing
pigmentation of the iris with that of the choroid and the retina in the same eye.
The iris grows out of the outer rim of the optic cup, beginning on about the
17th day of gestation, by which time the choroid and the retina are wellestablished structures. It has two layers, inner and outer, and both are densely
pigmented. The inner layer is essentially an extension of the pigmentary epithelium of the retina, from which it derives its pigment cells, but it forms pigment
considerably later than the retinal epithelium. The outer layer is essentially an
extension of the choroid, from which it derives its pigment cells, but it forms
pigment a little earlier than the choroid. Thus, the melanocytes of the iris,
although closely related to those of the retina and the choroid, differ from them
not only in their place of abode but also in their time of function. As this could
well mean differences in tissue environment, it was thought that clear and
consistent differences in the pigmentation of these structures would constitute
evidence for the role of the tissue environment.
MATERIAL AND METHODS
This report is based on an examination of the eyes of 69 mice belonging to
eight genotypical classes: seven were normal ( + / +), eight + fmi, thirteen Miwllj +,
eleven Miwhjmi, six MiwhIMiwh, thirteen s/s, five Wv/+ and six WvfWv. The
genetic background was heterogeneous, the intention being to obtain the widest
range of the effects of the gene concerned. The age of the animals varied from
2 weeks to 6 months. A part of this material had been used in another study
(Deol, 1971).
Expression of spotting genes in the mouse
485
Fig. 1. Retina (arrow) and choroid from a normal ( + / + ) mouse in transverse
section. Both structures are pigmented.
Fig. 2. Iris from the same eye as in Fig. 1. Both layers are pigmented, and so
indistinguishable from each other.
Fig. 3. Retina and choroid from a Mi'ch/+ mouse. The choroid (arrow) is not
pigmented.
Fig. 4. Iris from the same eye as in Fig. 3. Both layers are pigmented, and so
indistinguishable from each other.
Fig. 5. Retina (arrow) and choroid from a Miwhjmi mouse. Neither structure is
pigmented.
Fig. 6. Iris from the same eye as in Fig. 5. Only the inner layer (upper one here),
which corresponds to the retinal epithelium, is pigmented.
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M. S. DEOL
Some of the eyes were fixed in Bouin's fluid after enucleation. The lens was
removed and the eyes embedded in paraffin. They were sectioned at 10 /im,
and stained with Ehrlich's haematoxylin and eosin. Others were fixed in situ in
Witmaack's fluid, double-embedded in celloidin and paraffin, sectioned at 10 jtim
along with the surrounding structures, and stained in the same way.
OBSERVATIONS
The pigmentation of the choroid in many of these genotypes has been
described before (Deol, 1971), but will be summarized again here. The genes at
the mi locus also affect the structure of the eye, and the eyes of Miu:h/mi and
Miw1>/Miwh mice are abnormal in several ways, but this aspect of these genotypes
will not be considered here, because it is not pertinent to the purpose of this
study.
In normal ( + / + ) mice the choroid, the retina and both layers of the iris
were densely pigmented (Figs. 1, 2).
In 4-1 mi mice the choroid had large unpigmented regions or ' spots', but the
retina was fully pigmented. The iris was wholly pigmented in both layers, the
choroidal' spots' stopping short of it.
In Miwhf+ mice the choroid was generally very unevenly pigmented: in some
parts the density of the melanocytes was greatly reduced, while in others no
pigmented cells could be seen (Fig. 3). There were no normally pigmented
regions. In some animals the entire choroid was unpigmented. The retinal
epithelium had a good deal of pigment throughout, probably not much less
than in normal mice. The iris was fully pigmented in most of the animals
(Fig. 4), but in some, especially those with no pigment in the choroid, the intensity of pigmentation was reduced in the outer layer. This was particularly true
of younger animals. In all cases, however, the density of pigment in the outer
layer of the iris was much greater than in the choroid of the same eye.
In Miwhjmi mice both the choroid and the retinal epithelium were unpigmented (Fig. 5). An occasional retinal cell had some pigment granules, but they
were usually mis-shapen and unevenly distributed. In the iris the outer layer
was unpigmented, but the inner layer always had some pigment, although it
was unevenly distributed and much below normal in density (Fig. 6).
In Miwh/Miwh mice the choroid was devoid of pigment. The pigmentary layer
of the retina was mostly missing, but it was unpigmented where present. The
outer layer of the iris was unpigmented, but the inner layer always had a little
pigment in some parts.
The appearance of all three structures was essentially the same in s/s and
Wvf + mice as in + /mi mice.
In WvjWv mice the choroid and the outer layer of the iris were unpigmented,
but the retina and the inner layer were fully pigmented. The outer layer occasionally had an odd melanocyte in it, but it was always clearly of retinal origin.
These observations are summarized in Table 1.
Expression
487
of spotting genes in the mouse
Table 1. Summary of the effects of the genes mi, Mi wh , s and Wv
on the pigmentation of the eye
Iris
Choroid
Retina
Outer layer
Pigmented
Pigmented
Pigmented
Mi"h/mi
Pigmented
Spotted
Mostly
unpigmented
Unpigmented
Unpigmented
Pigmented
Pigmented
Mostly
pigmented
Unpigmented
Mi"'n/Mi"'h
Unpigmented
Unpigmented
Unpigmented
s/s
Wv / +
Spotted
Spotted
Unpigmented
Pigmented
Pigmented
Pigmented
Pigmented
Pigmented
Unpigmented
+/+
+ //Ml
wh
Mi / +
WrjWv
Inner layer
Pigmented
Pigmented
Pigmented
Mostly
pigmented
Partly
pigmented
Pigmented
Pigmented
Pigmented
DISCUSSION
The foregoing observations cannot be easily explained except by assigning
an important role to the tissue environment in the expression of genes at all
three loci. This is clearest of all in regard to the mi locus. In MiwhJ + mice the
pigment cells in the choroid form little or no pigment, but their kindred cells in
the outer layer of the iris in the same eye form considerable amounts, enough to
give it a normal appearance in most cases (Figs. 3, 4). There is evidently something in the tissue environment of the outer layer, presumably some melanogenesis promoting factor or factors, which brings about a change in the
behaviour of these cells. Similarly, in Miwh/mi mice the melanocytes in the
pigmentary layer of the retina remain unpigmented, except for an occasional
cell here and there, but their derivatives in the inner layer of the iris form appreciable quantities of pigment. Again, the same explanation appears hard to
escape. As to the genotypes +/mi, s/s, and Wv/ + , the situation is essentially
the same as in Miwh/+ mice, for the choroidal spots do not extend into the
outer layer of the iris. In Wv/Wv mice the choroidal melanocytes are presumably
so abnormal that they cannot differentiate in the iris either.
To sum up, the complete spotting patterns (both external and internal) of the
mutants considered here may be assumed to be the products of genetic abnormalities of pigment cells and normal variations in the distribution of some
melanogenesis promoting factor or factors in the host tissues. It is possible that
pigment cells are not uniformly affected throughout the animal, but it does not
appear essential to make this assumption. When spotting in an organ does not
follow any obvious pattern, as happens in the Harderian gland in these mutants
(Deol, 1971), it may simply mean that its pigment cells have reacted to melanogenic stimuli that they received in some other tissue during their passage through
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M. S. DEOL
it, in addition to those received in the tissue of their destination. Incidentally,
the mechanism of action of spotting genes, as sketched here, is exactly the
reverse of that of the genes at the Agouti (A) locus. The pigmentation patterns
produced by genes at the A locus also result from an interaction between the
pigment cells and their tissue environment, but the site of gene action here is
the host tissue, not the pigment cells (Silvers & Russell, 1955; Mayer & Fishbane, 1972).
The widespread assumption that the pigment cells which fail to differentiate
do not survive does not appear to be well founded. At any rate, it is not invariably correct. Some grounds for doubt have been given before (Deol, 1967,
1971), but the most unambiguous evidence against it comes from the retinal
melanocytes in Miwh/mi mice. Here the identification of the amelanotic pigment
cells is beyond doubt because of their regular position and arrangement (Fig. 5).
In this genotype very few of these cells form any pigment, yet they are all there,
forming a continuous epithelium. Could not the same be true of the migratory
melanocytes as well, the difficulties in the identification of amelanotic migratory
cells being purely technical? Migratory melanocytes are recognized by their
characteristic shape and the presence of melanosomes, but if along with their
capacity to form melanosomes they have also lost their distinctive shape, they
will be hard to identify. It is of interest that the firmest statement of the
view that undifferentiated pigment cells die is also based largely on studies
with genes at the mi locus (Mintz, 1971).
ZUSAMMENFASSUNG
Gene, die Weissfleckung in der Maus verursachen, wirken hauptsachlich auf zwei Weisen:
manche wirken auf die Melanoblasten, wahrend andere ihre Wirkung auf die Gewebeumwelt
der Melanoblasten ausiiben. Die Frage wurde gestellt, ob die normale Gewebeumwelt bei
der Herkunft der Fleckung in jenen Mutanten eine Rolle spielt, in denen angenommen wird,
dass die Melanoblasten der Ort der Gentatigkeit sind. Eine fruhere Untersuchung in der
Genotypen +/mi, Miwh/+, sis, Wv/+ and WV\WV wendet wurden, deutete darauf hin da
hochst prezise Pigmentierungsmuster auftreten. Es schien, als ob eindirekterBeweisgefunden
werden konnte indem die Pigmentierung der Iris mit der der Retina und dem. Choroid im
gleichen Auge verglichen wurde in diesen und anderen Genotypen. Die inneren Schichten
der Iris erhalten ihre Pigmentzellen von der Retina, die ausseren vom Choroid. Folglich
wurde ein ausgepragter und standiger Unterschied zwischen dem Verhalten dieser Zellen in
ihrem ursprunglichen und sekundaren Funktionsort Beweis sein fur die wichtige Rolle die
die Gewebeumwelt spielt. Diese Unterschiede wurden gefunden.
Ausserdem wurde in einem weiteren Genotyp (Miwhlmi) gefunden, dass die Pigmentzellen
der Retina deutlich zu unterscheiden sind, obwohl sie nicht pigmentiert sind. Dies macht die
weitverbreitete Annahme, dass Melanoblasten, die sich nicht differenzieren, absterben,
zweifelhaft.
The author is most grateful to Mr D. J. Patterson and Miss Gillian Skinner for
technical assistance.
Expression of spotting genes in the mouse
489
REFERENCES
BILLINGHAM,
R. E. & SILVERS, W. K. (1960). The melanocytes of mammals. A. Rev. Biol. 35,
1-40.
DEOL, M. S. (1967). The neural crest and the acoustic ganglion. /. Embryol. exp. Morph. 17,
533-541.
DEOL, M. S. (1971). Spotting genes and internal pigmentation patterns in the mouse. /.
Embryol. exp. Morph. 26, 123-133.
MARKERT, C. L. & SILVERS, W. K. (1956). The effects of genotype and cell environment on
melanoblast differentiation in the house mouse. Genetics, Princeton 41, 429-450.
MAYER, T. C. (1965). The development of piebald spotting in mice. Devi Biol. 11, 319-334.
MAYER, T. C. (1967a). Pigment cell migration in piebald mice. Devi Biol. 15, 521-535.
MAYER, T. C. (19676). Temporal skin factors influencing the development of melanoblasts in
piebald mice. J. exp. Zoo). 166, 397-404.
MAYER, T. C. (1970). A comparison of pigment cell development in albino, steel and
dominant-spotting mutant mouse embryos. Devi Biol. 23, 297-309.
MAYER, T. C. (1973). Site of gene action in steel mice: analysis of the pigment defect by
mesoderm-ectoderm recombinations. J. exp. Zool. 184, 345-352.
MAYER, T. C. & FJSHBANE, J. L. (1972). Mesoderm ectoderm interaction in the production
of the agouti pigmentation pattern in mice. Genetics, Princeton 71, 297-303.
MAYER, T. C. & GREEN, M. C. (1968). An experimental analysis of the pigment defect caused
by mutations at the Wand SI loci in mice. Devi Biol. 18, 62-75.
MAYER, T. C. & MALTBY, E. L. (1964). An experimental investigation of pattern development
in lethal spotting and belted mouse embryos. Devi Biol. 9, 269-286.
MINTZ, B. (1967). Gene control of mammalian pigmentary differentiation. I. Clonal origin
of melanocytes. Proc. natn. Acad. Sci. U.S.A. 58, 344-351.
MINTZ, B. (1971). Clonal basis of mammalian differentiation. In Control Mechanisms of
Growth and Differentiation, Symp. Soc. exp. Biol. 25, 345-369.
SEARLE, A. G. (1968). Comparative Genetics of Coat Colour in Mammals. London: Academic
Press/Logos Press.
SILVERS, W. K. (1956). Pigment cells: occurrence in hair follicles. /. Morph. 99, 41-56.
SILVERS, W. K. & RUSSELL, E. S. (1955). An experimental approach to action of genes at the
agouti locus in the mouse. /. exp. Zool. 130, 199-220.
{Received 29 January 1973)