The role of the tissue environment in the expression of spotting genes in the mouse

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, 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 melano-blasts 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 melano-blasts 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 well-established 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.

This report is based on an examination of the eyes of 69 mice belonging to eight genotypical classes : seven were normal ( + / + ), eight + /mi, thirteen Mi^wh/ +, eleven Mi^wh/mi, six Mi^wh/Mi^wh thirteen s/s, five W^v/+ and six W^v/W^v. 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). 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/m, 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 μm along with the surrounding structures, and stained in the same way.

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 Mi^wh/mi and Mi^wh/Mi^wh 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 +/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 Mi^wh/+ - 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 Mi^wh/mi 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 Mi^wh/Mi^wh 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 W^v /+ mice as in + /mi mice.

In W^v/W^v 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.

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 Mi^wh/ + 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 melano-genesis promoting factor or factors, which brings about a change in the behaviour of these cells. Similarly, in Mi^wh/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 W^v/ +, the situation is essentially the same as in Mi^wh/+ mice, for the choroidal spots do not extend into the outer layer of the iris. In W^v/W^v 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 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 in-variably 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 Mi^wh/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).

Gene, die Weissfleckung in der Maus verursachen, wirken hauptsächlich auf zwei Weisen: manche wirken auf die Melanoblasten, während andere ihre Wirkung auf die Gewebeumwelt der Melanoblasten ausüben. 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 Gentätigkeit sind. Eine frühere Untersuchung in der Genotypen +/mi, Mi^wh/+,s/s W^v/+ and W^v/W^v 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 äusseren vom Choroid. Folglich würde ein ausgepr ä gter und stä ndiger Unterschied zwischen dem Verhalten dieser Zellen in ihrem ursprünglichen und sekundären Funktionsort Beweis sein für die wichtige Rolle die die Gewebeumwelt spielt. Diese Unterschiede wurden gefunden.

Ausserdem wurde in einem weiteren Genotyp (Mi^wh/mi) 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.