modification of pigmentation patterns in allophenic mice by the w gene

Differentiation 9, 19-27 (1977) Differentiation 0 by Springer-Verlag 1977

Modification of Pigmentation Patterns in Allophenic Mice by the W Gene Jon Gordon Department of Biology, Yale University, New Haven, Ct. 06520, USA

Summary. Mouse embryos heterozygous at the W locus were combined with embryos which were wild type at this locus but homozygous for albino. The resulting allophenics displayed an unusual pigmentation phenotype consisting of entirely white fur and ruby-coloured eyes. Microscopic examination showed the eye pigment to be located exclusively in the retinal epithelium. which was a mosaic of black and white sectors. This ruby-eyed white pattern corresponds to what would have been expected for WWCC tt wwcc mosaics but not for WwCC c) wwcc mice. WW mice are black-eyed whites, but Ww mice have black eyes and black fur, except for a small ventral white spot. These results suggest that melanocytes of the Ww genotype, although capable of produc- ing normally pigmented fur in Ww animals, fail to populate hair follicles when in competition with wwcc (albino) melanocytes that are wild type at the W locus. The genotype of these WwCC c) wwcc allophenes was proved by progeny testing. This is apparently the first report of a single gene change affecting the competitive ability of cells in allophenic mice, and suggests that such changes may play a significant role in the clonal selection of embryonic cells during development.

Introduction

Each tissue of an adult mammalian organism must inevitably arise from a small number of embryonic precursor cells which are derived from a larger pool of poten- tial contributors. The basis for selecting certain cells and by-passing others, however, is not understood, and its elucidation is a fundamental problem in developmental biology. Previous workers have demonstrated [ 1,21 that the earliest recognizable step in mammalian cell differen-

tiation, that of inner cell mass vs. trophoblast determination, is based upon the position of each blastomere in the cleaving embryo. Those cells which find themselves on the periphery of the spherical morula become trophoblast cells, while those in the central zone become the inner cell mass. Later in development, however, when millions of cells are dividing and migrating, and the structure of the embryo is exceedingly complex, the position of a cell at a given moment in time is not sufficient to explain its developmental fate. Its previous history is also critical. One possible hypothesis is that during many cell divisions subtle genetic changes occur [3-51 which predispose cells to differentiate into specific tissue types, but their qualifications for such differentiation may vary somewhat. Thus a form of “natural selection” may be working, with the cells best able to multiply and differentiate in a particular direction eventually compos- ing the adult tissue. Such a hypothesis has been difficult to test, however, since all of the cells in the embryo are in a gross sense genetically identical and the number of precursor cells required for each adult organ is still un- known. These difficulties are compounded in mammals, where the embryo is sequestered in the uterine environment, inaccessible to experimentation.

The successful production of allophenic mice, [6, 71 however, now permits the creation of chimeric embryos made up of cells with different but distinct genetic markers. The adult tissues of these mosaic mice can be examined, and the proportion of cells of each genotype mea- sured. Consistent predominance of one cell genotype over the other in a given tissue of a number of allophenics would suggest that the genetic makeup of this cellular component gave it a selective advantage in forming that tissue.

Such patterns of dominance have in fact been observed in chimeras. Tuffrey et al. [81 have shown that in

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AKR c) CBA mosaics, the lymphocyte population is consistently dominated by the AKR strain. In C57B1/6 ++ C3H chimeras 191, the C57 cells dominated in the bones of the skull, while the vertebrae in the lumbosacral region were populated largely by C3H cells. A number of allophenic combinations also result in dominance in the germ line of one strain over the other [lo]. The genetic composition of cells has thus proved to be impor- tant in the competitive ability of cells in the chimeric embryo.

Attempts to assign this type of competitive advantage to a specific gene, however, have been less successful. Combining embryos homozygous for retinal degeneration (rd) with normal embryos resulted in large por- tions of the mosaic retinas being populated by rd/rd cells 111, 121, though more recent experiments have shown some dominance of +/+ cells [131. Mosaics composed of normal cells and cells homozygous for the mutations vestigial-tail and short-ear, both of which produce skeletal abnormalities, resulted in allophenics with moderate skeletal defects 1141, showing that the mutant cells gained access to the skeleton and were not competed out by normal cells. Thus, to date, no specific allele has been identified which affects the ability of an embryonic cell to compete with cells not containing that allele.

This paper reports the identification of such a gene - the W gene. This gene manifests pleiotropic effects on blood cells [151, pigment cells 1161, and germ cells 1171. The WW homozygote suffers from lethal macrocytic anaemia. When rescued by a bone marrow transplant, the surviving animal eventually displays a black-eyed white phenotype. The white areas are devoid of recognizable melanocytes [lBI. The WW skin has been shown to permit the differentiation of grafted melanocytes [ 191, but the restriction of pigment in spotting mutants has nevertheless been shown to be due at least in part to the tissue environment [201. Probably, spotting patterns are produced by multiple influences, operating both inside and outside the melanocyte. Pigment in the eyes of WW animals is due to a normally pigmented retinal epithelium, a tissue unaffected by the W gene. The choroid layer of the eye, normally populated by pigment cells derived from the neural crest, is also devoid of pigment in WW mice, showing that the affect of the W locus on pigment is confined to melanocytes derived from the neural crest. The W/+ mouse is normally pigmented except for a white ventral spot and white feet and tail tip (Figs. 1 and 2).

We produced allophenic mice from such embryos combined with embryos carrying the wild-type allele at the W locus but homozygous for albino - that is,

J. Gordon: W/+ Melanocytes in Mosaic Mice

wwcc. The WwCC c) wwcc allophenics produced had ruby-coloured eyes and white fur. Histological examination of the eyes of these mice showed that all pigment was confined to the retinal epithelium, which was a mosaic of pigmented and white areas. Control wwCC ++

wwcc embryos fused in these same experiments and therefore, on the same genetic backgrounds, had exten- sive pigment in the fur and showed mosaicism in both the choroid layer and retinal epithelium of the eyes. The genotypes of the allophenics were proved by progeny testing. These results clearly show that although Ww pigment cells readily differentiate in hair follicles of the Ww mouse, these same pigment cells fail to populate hair follicles in the presence of normal pigment cells in WwCC c) wwcc allophenics. Thus a single allele dici- sively affects the competitive ability of melanoblasts in the developing allophenic embryo.

Methods

Production of Allophenic Mice

C57B1/6-Ww, C57B 1/6-WVw, and B6D2F, mice were obtained from the Jackson Laboratories, Bar Harbor, Maine. Swiss ICR albino mice were obtained from the Charles River Breeding Laborato- ries, Wilmington, Mass. Prior to use, the C57B1/6-Ww mice were crossed to B6D2F1 mice. The offspring which carried the W allele were designated HYB-Ww and were used for production of WWCC, WwCC, and wwCC embryos. Swiss albino mice were either used directly or first crossed with B6D2F1 mice; the offspring were subse- quently mated with each other and the albino progeny selected for production of chimeras. This albino strain was designated HYB- Al.

Embryos carrying the W gene were obtained by mating B6D2F1 or HYB-Ww females with HYB-Ww males. The HYB-Ww x HYB- Ww cross produced WWCC, WwCC, and wwCC embryos in a ratio of 1 : 2 : 1. These embryos were fused with ICR embryos. The B6D2F1 x HYB-Ww cross produced WwCC and wwCC embryos in equal numbers. These were fused with HYB-A1 embryos.

Allophenic mice were produced by the method of Mintz et al. [211, with the modifications previously described [22].

Histological Preparations

Whole eyes were removed under anaesthesia from control WvWCC, WwCC, and wwCC mice, and from experimental chimeras and placed directly into Bouin's fvtative (W homozygotes were produced by mating C57B1/6-Wvw parents. The W" mutation is an allele of W which is viable in the homozygous condition. WvWv mice have the same pigmentation pattern as WW mice but are easier to produce). After 48 h in Bouin's fixative, the eyes were cut into equal halves along the caudo-cephalic axis and the lenses removed. The remaining tissue was dehydrated in ETOH, cleared in xylene, and embedded in 52-54O tissue mat. Nine micron sections were cut, cleared of paraf- fin, and mounted without staining in permount.


Fig. 1. Dorsal view of WwCC (right) and wwCC (left) mice. Note the heavy pigmentation of both mice. Arrows denote beginning of unpigmented areas of the WwCC mouse

Fig. 2. Ventral view of WwCC (right) and wwCC (left) mice. The ventral white spot is clearly seen on the WwCC mouse

Fig. 3. View through a dissecting microscope of the eye of a WwCC tf wwcc allophenic mouse. Pigmented and unpigmented zones can be easily distinguished. The pigment was found on microscopic examination to be located entirely in the retinal epithelium

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22 J. Gordon: W/+ Melanocytes in Mosaic Mice

meras. These mosaics resulted from fusions in which 25% of the pigmented embryos were expected to be homozygous for the W gene. A WWCC H wwcc chimera would be expected to have completely white fur, since both component genotypes are themselves white. The retinas of such chimeras would be expected to be a mosaic of pigmented and albino cells, since WW mice have fully pigmented retinas. Thus, the pigment phenotype of this chimera should be the same as that expected of a WwCC tt wwcc allophenic, in which all of the neural crest-derived melanocytes from the Ww component were excluded from hair follicles by the ww melanoblasts. This expectation can in principle be demonstrated by breeding the mosaic mice. A WW mouse does not produce germ cells, while a Ww mouse is normally fertile. Thus, a WwCC c) wwcc mosaic, when mated to an albino mouse, might well be expected to

Progeny Testing of Chimeras

The genotypes of the allophenics were determined by mating them with HYB-A1 albinos of the opposite sex. Albino offspring indicated the presence of albino cells in the germ line of the allophenics. The pigmented offspring were identified as carrying the W gene by the presence of a ventral white spot and by white extremities. Only those offspring showing an unmistakable ventral white spot were assumed to be Ww. The presence of Ww progeny proved that the parent chimera contained Ww cells. The absence of Ww offspring among the pigmented progeny proved that the parent's pigmented component was due to ww cells-that is, wild-type cells.

Results

Twenty-four allophenic mice in this series have thus far been produced. Table 1 shows the sex phenotype, pigmentation pattern, and mating records of 13 of the chi-

Table 1. Allophenics resulting from (HYB-Ww x HYB-Ww) - (ICR x ICR) fusions

Mouse Sex % Pigment % Pigment Albino Pigmented progeny No. in coat in eyes progeny -

ww w w

80 81 82 83 84 85 86 87 88 89 90 91 92

M 0 F O* F 0 M O* M 0 M 50 F 15 F O* F 40 M 0' F O* M 0 F 85

25 50 80 50 10 40 60 80 30

100 50 80 10

54 34 63

113 87 85 70 54 33 0

57 60 45

0 0 3 0 0 0 0 0 0 0 0 0 3 ___

0 0 2 0 0 0 0 0 0 0 Sterile 0 0 0

Table 2. Allophenics resulting from (HYB-Ww x B6D2F,) - (HYB-AI x HBY-A1) fusions

Mouse Sex % Pigment % Pigment Albino Pigmented progeny No. in coat in eyes progeny

ww w w

93 94 95 96 97 98 99

100 102 103 104

-

M F F M M M M M F F F

5 95 0 0 0 0 1

40 35 0 0

50 99 + 50 10 90 70 90 45

5 15 5

__ 52 1 1 4 0

35 7

29 1 1 1 1 11 16

~

4 4 12 0 3 1

22 11 0 0

13 20 1 0 0 0 0 0 0 0 0 0


Fig. 4. Section of the eye of a W"W homozygote. Pigment is located in the retinal epithelium only. Compare with Figure 5, where both layers are pigmented. x 1000

Fig. 5. Section of the eye of a WwCC mouse. Note that both the choroid (below) and the upper retinal layer are fully pigmented. Arrow shows the division of the pigmented layers. x 1000

Fig. 6. Section of the eye of a WWCC tf wwcc chimera. Gaps in pigmentation are areas occupied by the albino (wwcc) cells. Arrow shows a portion of the eye in which the choroid alone is pigmented. The unpigmented retinal cells can be clearly seen. In the patch of pigment furthest to the lefl, both layers are pigmented. x 400

Fig. 7. Section of the eye of a WwCC u wwcc allophenic mouse. Gaps in pigment are areas occupied by albino (wwcc) cells. Note that all pigment is located in the retinal layer. The strip of tissue was twisted on itself during preparation, so that the pigmented retina appears at the upper right to be facing away from the rods and cones, which can be seen at the upper I&. Arrows show the unpigmented choroid layer. x 160

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produce pigmented offspring, half of which would be WwCc, and the other half wwCc. A WWCC tf wwcc chimera would be expected to produce only albino offspring. Examination of Table 1 shows that most of the chimeras indeed produced exclusively albino offspring. Mouse # 82, however, despite having completely white fur, produced both WwCc and wwcc progeny. This result proves that this mouse contained WwCC and not WWCC cells. Mouse # 89 was sterile and, as shown in Table 1 had no albino pigment cells in the eyes. This mouse was also shown to have macrocytic anaemia, and no evidence for chimerism was observed. Many of these mice, including # 89, had extremely small patches of pigmented fur, designated in Table 1 by the “O*” symbol. In mice #s 81, 83, and 89, this spot was located near the dorsal midline, but in mice #s 87 and 90, the spot was located at some distance from the dorsal midline. In mouse # 87, the spot was located at the tip of the right hind leg, a location normally unpigmented in both Ww and WW mice.

The second group of mosaics, listed in Table 2, resulted from fusions in which the pigmented component of the chimeras could only have been Ww or ww. Mice #s 95, 96, 97, 98, and 103 all had completely white coats and substantial pigmentation in the eyes. Mouse # 93 had a small amount of fur pigment. Mice #s 93, 95, 96, and 98 all produced both WwCc and WWCC offspring, proving their genotypes to be WwCC ++

wwcc. Mice #s 94, 100, and 102 all showed substantial amounts of fur pigment. Mouse # 9 4 was proven by breeding to be wwCC tf wwcc, but the other mosaics with significant amounts of fur pigment produced no pigmented progeny, and thus their genotypes could not be deduced from their progeny.

When examined under the dissecting microscope, the eyes of all ruby-eyed white mice displayed a pigmentation pattern similar to that shown in Figure 3. In con- trast to the fur, the eyes were heavily pigmented. The existence of white areas in the eyes proved that these mice were true chimeras.

The retinas of control W W V and Ww mice were examined histologically, and the results are shown in Figures 4 and 5. The WvWv mouse had no pigment cells in the choroid layer, while the retinal epithelium was fully pigmented. The Ww mouse was fully pigmented in both layers of the eye. A histological section of the eye of a wwCC cs wwcc (# 94) is shown in Figure 6 . The choroid and retinal layers both contain patches of pigment, the result expected for a wwCC ++ wwcc chimera. All eyes examined from allophenics having completely white fur were similar to the result shown in Figure 7. The choroid coat was completely unpigmented, while


the pigmented retina was patchy. All ruby-eyed white allophenes examined in this fashion had been proved by breeding to be WwCC c, wwcc mosaics.

Discussion

The results presented here clearly demonstrate that Ww pigment cells originating from the neural crest are un- able to compete with ww melanoblasts in allophenic mice, despite the fact that the Ww mouse has large numbers of these cells. Furthermore, this failure to compete could not have been due to the strains of mice employed in these fusions, since wwCC tf wwcc mice produced in the same experiments, and therefore on the same genetic backgrounds, were heavily pigmented. Histologic examination of the pigmented layers of the eyes revealed that this competitive advantage of ww cells prevailed in those eye tissues populated by dendritic melanoblasts just as in the fur. The pigmented retina which is populated by pigment cells of a different embryonic origin was not affected. Thus the dominance of ww cells or Ww was not a generalized phenomenon affecting all pigment cells, but was confined specifically to neural crest derived pigment cells.

These data do not prove that all WwCC ct wwcc chimeras show this ruby-eyed white phenotype. Mice #s 80,8 1,83,84,87,90, and 9 1 in Table 1 all produced albino offspring only. Thus, some of them could have been wwCC tf wwcc mice which for one reason or another produced no germ cells from the wwcc component. Moreover, mouse # 92 had heavily pigmented fur and did not produce enough pigmented offspring to rule out the possibility that it was WwCC c) wwcc. The fact that none of its progeny carried the W gene, however, suggested strongly that this mouse was in fact wwCC ++

It should be recognized that in this type of experi- ment there will always be allophenics produced which cannot be progeny-tested in the desired fashion. Some will inevitably be sex mosaics, in which the parental strain of interest will have the opposite sex genotype from the phenotype of the allophene. Since no germ cells will arise from XX cells in a male mosaic or from XY cells in a female [lo, 23, 241 the exact genotype of these cells cannot be determined by progeny testing.

Sex mosaicism is undoubtedly part of the explanation for the failure of so many of the allophenes listed in Table 1 to produce pigmented offspring, but it cannot be the only explanation. None of the mice listed in Table 1 produced exclusively pigmented offspring, which would have been the case for a sex mosaic mouse in which the

wwcc.


sex genotype of the pigmented cells corresponded to the phenotype of the animal. This latter situation should occur 50% of the time.

One explanation of this discrepancy is that all of the ruby-eyed white mice produced in this first group were WWCC c) wwcc mosaics. In this case the WW genotype would be expected to eliminate the germ cells and the progeny would thus all be albino. This explanation is unlikely, however, since WWCC t) wwcc allophenics would be expected to arise only 25% of the time rather than at the 11 out of 13 rate observed in this group.

Another possibility is that the ICR primordial germ cells in these chimeras had a competitive advantage over the HYB-Ww strain, a phenomenon which has been previously reported for germ cells in allophenic mice [lo]. This explanation is supported by the results presented in Table 2, where a different albino strain was used. This table shows that 6 of the 11 mice produced passed on germ cells from the pigmented strain, and one of them, mouse #96, passed on germ cells from Ww CC line only. The HYB-Ww germ cells thus appear to compete better with the HYB-A1 cells than with ICR cells.

A third and intriguing possibility is that Ww germ cells are at a competitive disadvantage by virtue of the fact that they carry the W gene. Since the WW genotype is fatal to germ cells, as it is to dendritic pigment cells, the possibility arises that in a Ww c) ww mosaic, one copy of the W gene affects the competitive ability of primordial germ cells just as it does for melanoblasts. The increased number of Ww H ww mice passing on germ cells from the Ww component in the second group of mice argues against this possibility, but does not rule it out. Mice #s 97, 103, and 104 are likely to be WwCC c1 wwcc mosaics since they have no fur pigment, but none of them produced pigmented offspring. Though all three mice may have been sex mosaics in which the HYB-A1 strain corresponded in genotype to the sex phenotype of the allophene, 103 and 104 are both females, and previous investigations have reported that very few sex mosaics become females [6, 251. Thus the possibility remains that 103 and 104 were XX c) XX mosaics in which the W gene caused the germ cells carrying it to be competed out during embryogenesis, One must keep in mind, however, that many mosaic combinations produce as many female sex mosaics as males [261, and the mosaics produced in this second group may represent such a combination.

The small flecks of fur pigment in many of the allophenics listed in Table 1 are of some interest. W'W' mice occasionally show small spots of pigment [Russell, personal communication1 ; and since a few melanoblasts normally escape the effect of the W gene, it is not sur-

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prising that similar markings appear in these allophenics. We therefore feel that these spots do not represent survival of pigment cells in significantly greater numbers than normally observed in WW mice, and thus do not contradict the conclusion that Ww pigment cells are being selectively excluded from hair follicles in WwCC t) wwcc mice. This assertion is supported by the fact that these tiny spots were never observed in mice proved to be WWCC c) wwcc. These latter allophenes were always heavily pigmented, showing a clear difference in pigmentation pattern between WwCC c) wwcc and WWCC c) wwcc mosaics. Though none of these flecks was seen in the second group of mosaics, it is our view that in fact Ww survived better in this series than in the series listed in Table 1. This opinion is supported by the observation that mouse # 93, which was proved progeny testing to be WwCC t) wwcc, had about 5% of the fur pigmented. A similar but smaller zone of pigment was observed in mouse # 99, which has not yet sired any pigmented offspring, and therefore cannot be con- firmed to be WwCC ++ wwcc. These areas were both significantly larger than the small spots seen in #s 81, 83, 87, 89, and 90 of the first group.

The location of the pigmented spot in mouse # 87 is possibly of great significance in explaining the mecha- nism by which the Ww genotype produces spotting. This spot was located at the tip of the right hind foot, an area which is consistently devoid of pigment in Ww mice. The white spots in Ww mice are always in the regions most distal to the dorsal midline, raising the possibility that Ww melanoblasts are deficient in their ability to migrate. The ruby-eyed white pigment pattern in # 87 strongly suggests that it is a WwCC ++ wwcc chimera. The presence of Ww pigment cells in this distal region suggests that these cells can migrate normally. The explanation for the absence of pigment cells in these areas in Ww mice thus may not be due to melanoblast migra- tory insufficiency, but rather to the fact that Ww pigment cells are marginally viable. As the cells migrate dorsolaterally in Ww mice, cells more proximal to the dorsal midline may die, leaving empty spaces which are filled by backward migration of more distally located melanoblasts. In the WwCC H wwcc mouse, these empty spaces may be filled by laterally migrating albino cells from a neighbouring clone, thus permitting the WwCC cells to proceed to this distal location.

This postulated cell death is not analogous to the pre-programmed cell death proposed by Mintz 141 to explain the Ww phenotype. She produced two mosaics from WwBB x WwBB and wwbb x wwbb fusions which had a white belly spot surrounded exclusively by brown (bb) pigment. She concluded that these were

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WWBB t) wwbb allophenics and remarked that their phenotype resembled that of a Wwbb mouse. This re- semblance suggested to her that in Ww mice, pre-programmed cell death occurs among some of the melanoblasts distributed along the dorsal midline prior to the onset of migration, leaving ghost zones which had to be filled by melanoblasts from neighbouring zones. The la- teral migration of these neighbouring melanoblasts pre- cluded their distal migration, and resulted in the most distal areas being unpigmented. The spotting patterns in our allophenics suggests that cell death may not be the only effect of the Ww genotype. Our WwCC c) wwcc allophenics clearly show that fewer Ww melanoblasts occur at the dorsal midline than wwcc melanoblasts. This suggests that W may be working at an earlier point to impair the differentiation of melanoblasts. Thus, the Ww embryo may have fewer primordial melanoblasts initially lined up on the dorsal midline. If the pre-programmed cell death previously described was the only force operating on the Ww pigment cell, one would expect WwCC t) wwcc mosaics occasionally to show large bands of pigmented fur, a phenotype which we have not observed. Our results suggests that the two mice originally thought to be WWBB H wwbb in this prior report might well have been WwBB c) wwbb mice showing the same effects on pigmentation that we have observed in our study. The presence of macrocytic anaemia in one of these allophenes cannot be taken as strong corroboration of the WWBB c) wwbb genotype, since WW mice rescued neonatally by bone marrow transplants show no evidence of anaemia [271. Thus, one would expect that in a WW c) ww allophenic mouse the ww haematopoetic stem cells would replenish the empty sites left by the WW cells and a normal blood picture would then result. Of course, by chance all the haematopoetic stem cells in the chimeric embryo might have been WW cells, but then the allophene should have suffered a lethal macrocytic anaemia. This unusual mouse might have been a rare example of an allophene in which all of the haematopoetic stem cells were WW but which nevertheless resulted in a non-lethal anaemia, a phenomenon which has been known to occur [Russell, personal communicationl, and which may have also oc- curred in our mouse # 89. The major difference between our # 8 9 and the presumed WWBB c) wwbb mouse previously reported is that our mouse showed no evidence of chimerism, which suggests that no normal blood cells could have been present to rescue the mouse.

The discovery of a gene which selectively suppresses a specific cell type in chimeric mice should be useful in further studies of the developmental genetics of cellular


interactions. Other genes known to modify gene expression in the melanoblast, such as the gene for agouti [281, can be tested for their ability to promote the survival of Ww melanoblasts, and further to define the role of the tissue environment in the differentiation of melanoblasts. Other alleles of the W gene can be investigated for their comparative effects on melanoblasts and for their effects on the selection and survival of primokdial germ cells. These results also suggest that single allelic differences can affect not only the obvious phenotypic characteristics of cells, but also the ability of cells to compete with one another during morphogenesis of the embryo.

Acknowledgements: This research was supported by grants from the Ford Foundation, NIH Grant R01 HD 07741-03, and NSF Grant PCM76-23036 to Dr. C. L. Markert.

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modification of pigmentation patterns in allophenic mice by the w gene

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