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A comparison of pigment cell development in albino, steel, and dominant-spotting mutant mouse embryos
DEVELOPMENTAL
BIOLOGY
23, 297-309 (1970)
A Comparison Albino,
of Pigment
Cell Development
in
Steel, and Dominant-Spotting Mutant
Mouse
Embryos1
THOMAS C. MAYER Department
of Biology,
Rider College, Trenton,
New Jersey 08602
Accepted July 16, 1970 INTRODUCTION
Melanin-synthesizing cells found in the coat of the mouse originate from the neural crest and migrate throughout the skin during days 8-12 of embryonic development (Rawles, 1947). Thirty-four primordial melanoblasts apparently are set aside early in the development of the mouse embryo (prior to day 7) and are lined up longitudinally, 17 on each side, during the initial formation of the neural crest (Mintz, 1967; however cf. Lyon, 1968). As revealed through studies on the chick embryo (Rawles, 1944; Weston, 1963), melanoblasts then undergo rapid proliferation and migrate laterally to occupy the skin up to a certain density. The wave of migrating cells continues to move into skin regions not occupied by melanoblasts, and migration ceases when all areas are occupied to their maximum density (Willier, 1952). The melanoblasts in the skin produce melanin long after the migration process is completed, and the sequence of their maturation does not correspond to the time of arrival of melanoblasts in a specific region (Rawles, 1955; Mayer and Reams, 1962). This complex process of early organization, migration, and differentiation of the melanoblast component of the neural crest is under genetic control, with the two obvious sites of control being the skin or the melanoblast itself. Disturbances in the organization of the primordial melanoblasts, their proliferation, migration, survival, or differentiation can result in defects or gaps in the coloration of the skin and hair. This report represents an analysis of the action of genes at three distinct genetic loci, albino (c), steel (Sl) and dominant-spotting (IV). ’ This investigation Foundation.
was supported
by Grant 297
GB 7989 from the National
Science
298
MAYER
Mice homozygous for mutant alleles at the W-locus are characterized by severe macrocytic anemia, lack of hair pigmentation and sterility (Russell, 1954). The pigmentation defect has been analyzed initially through the use of a grafting technique in which neural crest from normal or mutant mice was combined with skin from the appropriate genotype. The site of action of W appeared to be the neural crest. Skin of the mutant genotype did not influence normal melanoblasts in any adverse way (Mayer and Green, 1968). The mutant gene Sl is a very close mimic of W. Steel homozygotes are black-eyed white, anemic, and sterile (Bennett, 1956). This similarity, however, is a superficial one for the two genes appear to produce the pigment defect through different sites. Combination grafting has revealed that the skin of SlISl mice plays the determining role in the pigmentation block. The melanoblast component of the neural crest is normal in steel embryos (Mayer and Green, 1968). The action of the albino gene is apparently simpler and certainly better understood than the spotting genes. There are no obvious pleiotropic effects associated with c, and the pigmentation defect appears to be through the melanocyte at the level of the enzyme tyrosinase. Albino mice possess clear cells in the matrix of the hair follicles where melanocytes normally are located (Silvers, 1956). These clear cells are thought to be amelanotic melanocytes incapable of synthesizing melanin. In addition, tyrosinase activity is lacking in the skin of c/c mice, and is reduced in c/+ genotypes (Coleman, 1962). The results being presented in this report are a further characterization of the mechanism of action of the W, Sl, and c genes with respect to the pigmentation defect. The only previous experimental study on W and Sl mutant embryos utilized skin from 11-day embryos. Since at this age it was impossible to identify the genotype of the mutant embryos produced from matings of heterozygotes, the results were based on a small statistical sampling of the embryos produced. The differences revealed in this previous work between the action of Sl and W, and particularly the effect of SlISl skin on normal melanoblasts, made it essential that this work be repeated using embryos of known genotype. In addition to a confirmation of these previous results, it was anticipated that the use of whitespotted skin following the presumed migration of its own melanoblast population might yield information regarding the status of this population. A specific time of action of the steel inhibitory effect could also be assessed by using skin from embryos of progressively
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older ages. This study, therefore, was an extension of the previous report, and utilized skin from 13-day and older embryos, ages at which the genotype of the embryo could be accurately determined. The albino was included in this study in order to compare the behavior of skin with its own complement of amelanotic melanoblasts to that of white-spotted skin. MATERIALS
AND METHODS
Embryos used as skin donors in the W grafting series were produced from matings between C57BL/6J - W I+ parents. The W’IW embryos could be separated from their W’/+ and +I+ littermates by the pale color of the liver at ages 13 days and older. Steel skin donor embryos were from mating C57BL/6J-Sld/+ parents, and these homozygotes could likewise be identified by their pale livers at 13 days. Embryos of the A/J strain (ala b/b c/c) were used as skin donors for the albino series. In all cases the source of normal melanoblasts was 9 days neural tubes derived from embryos produced by mating C57BL/6J - +/+ parents. The combination grafting method has been described previously (Mayer, 1965). Briefly, the neural tubes from the posterior quarter of g-day embryos were removed and carefully cleaned of all adhering somite and skin cells with the aid of trypsin digestion. Skin from the midflank between the limb buds of 13-day or older embryos was removed with finely ground iridectomy scissors and cut into 1 mm square pieces which were suitable for grafting. One neural tube and one piece of skin were then grafted together to the flank of 2.5 day White Leghorn chick embryos. After a 15-day incubation period, the grafts were located, dehydrated, and cleared in oil of wintergreen. The presence or absence of pigment was then tabulated from the cleared whole mounts of the grafts without the use of stains or other treatment. The areas examined for pigment were the hair follicles of the graft and the skin and coelomic lining of the host surrounding the graft site. Each series of experiments included a group of skin grafts implanted without associated neural tubes. These control grafts served as a verification of the genotype of the W/IV and SldlSld donor embryos, and as a check for any host pigment formation resulting from the operating procedure itself. RESULTS
A total of 266 operations were performed from which 222 grafts were recovered for study. Thirty operated embryos failed to survive the incubation period, and 14 host embryos were recovered in which
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a graft was not found. Extensive hair growth was present in all the grafts tabulated. The results of the three mutant series are summarized in Table 1. Albino
Series
The results of this series were divided into two groups. In the older group skin was used from embryos 13-18 days of age. This skin had received its own complement or invasion of melanoblasts prior to grafting. In the younger group, 11-day skin prior to receiving its melanoblast invasion was used. In both groups combination grafts were made using normal g-day neural tubes. Twenty-three grafts were recovered from implantations using skin of the older group, and twenty of these produced hairs that were totally free of pigment. Hair growth in grafts was extensive with typically 30 to 40 hair follicles associated with each transplant. Another constant feature of these grafts was the extensive development of pigment in the tissues of the host chick embryo immediately surrounding the graft. Melanocytes were observed extending out in all directions from the graft for a distance of up to 3 mm. Although the extent of melanocyte migration into the host tissues varied somewhat from one graft to another, the pigmentation of the host was a characteristic and obvious feature of this series. Figure 1 is an example of a typical graft of this type. The remaining three grafts each possessed a few pigmented hairs, although the majority were pigTABLE SUMMARY
OF THE COMBINATION ASSOCIATION
With +/+
GRAFTING
WITH NORMAL
1 RESULTS USING MUTANT NEURAL
SKIN IN
TUBES”
Skin-only
tubes
Type
Hair w/w skin (13-18 days) SldlSld skin (13-18 days) c/c skin ( 13-18 days) c/c skin ( 11 days)
Coelom
Hair
Coelom
+cw
+(28)
-(21)
-(21)
- (60)
- (58) +(5)
-(31)
- (31)
+ -(3)
-cm
+(X9
-08)
-(18)
+-(3) +(22)
+cm
-U6)
-0.3
’ The skin-only column represents control grafts. + = pigment present; - = pigment absent; + and - = grafts having both pigmented and pigment-free hairs. Numbers represent grafts recovered of each type.
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FIG. 1. Combination graft resulting from the inplantation of 13-day c/c embryonic skin with normal neural tube. Note the pigment-free graft and hair follicles with a trail of melanocytes away from the graft into the host tissues.
ment-free. In these three cases, pigment cell migration into the host tissues was comparable to that from those grafts which completely lacked pigment in the hair follicles. It appeared likely that the cause of the pigmentation failure within the hair follicles of these older skin grafts was the presence of amelanotic melanoblasts already in the skin and occupying the spaces available to other melanoblasts. In order to clarify this point, a young skin series was undertaken in which 11-day albino skin was combined with normal (+ /+) neural tubes. Skin of this age had not yet received its own melanoblast invasion, and therefore did not possess a population of amelanotic melanoblasts. Twenty-two grafts were recovered, and all possessed extensive hair development with all hairs pigmented. In addition melanocytes were found within the host tissues surrounding the grafted skin. It appeared, therefore, that the presence of an amelanotic melanoblast population in the skin of the older series was an effective block to the entrance of additional normal melanoblasts. In conjunction with both the old and young grafting series, a group of skin-only control grafts was made. The purpose of the control series was to determine with certainty the origin of the melanocytes found in association with the combination grafts. Eighteen skin-only implantations of the older series and 16 of the younger series were recovered. Extensive hair growth was observed in the grafts of both series, but pigment was absent from both the graft and the host tissues in the operated area. These results clearly demonstrated that the operative procedure had no effect on the occurrence of pigment
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in the host, and that the melanocytes observed in the region of the graft were derived from the donor neural tubes. Dominant-Spotting
Series
Embryos presumed to be of the W’lW’ genotype because of the pale liver color were selected as skin donors. As an additional check of the donor genotype, skin-only grafts were made from each embryo used in the combination grafts. One embryo was discovered to have been incorrectly classified, and it was discarded from the tabulation of the results. The control grafts from all the other presumed W’/W’ embryos failed to produce pigment. Pigment-free hairs in large numbers were present in each graft, and the host tissues surrounding the operated area were devoid of melanocytes. Combination grafts in this series were produced by implanting skin from 13-18-day W’IW’ embryos in conjunction with g-day normal neural tubes. The results of these combination grafts were remarkably consistent. Twentyeight grafts were recovered, and all possessed large numbers of pigmented hairs (Fig. 2). In no case were any pigment-free hair follicles observed in grafts of this combination. The tissues of the host surrounding the graft were occupied by melanocytes in numbers essentially similar to that of the previous series (Fig. 3). Steel Series Presumed Sld/Sld embryos were identified in the same way as the WV/W’ embryos, and skin-only grafts were made to verify the genotype. One embryo was incorrectly classified, and the error was apparent from the control series. Thirty-one skin-only grafts were recovered from Sld/Sld skin implantations, and all were devoid of pigment both in the graft and in the surrounding host tissues. Sixtythree grafts were recovered in this series from combining skin 13 to 18 days of age with g-day normal neural tubes. The large majority of these grafts produced only pigment-free hairs (Fig. 4). Hair growth from the SldlSld skin was extensive and the relationship of the grafted skin to the neural tube at the time of recovery was identical to the previous two series. The absence of pigment from the steel hair follicles was not absolute, for in a few grafts some pigmented hairs were found along with those lacking pigment (Fig. 5). Only three cases of these mixed grafts were found from the total of 63 recovered cases. The occurrence of melanocytes in the host tissues was also of particular interest in this series. In only five cases were
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FIG. 2. Combination graft of 13 day W‘/W’skin with normal neural tube. Intense pigmentation of hair follicles is obvious. FIG. 3. Combination graft of 13 day W /W skin with normal neural tube. Migration of melanocytes into host tissue is extensive.
melanocytes found in the host tissues surrounding the graft. The area of the host immediately adjacent to the remaining 58 grafts of this series was completely devoid of melanocytes. This finding is particularly interesting when compared to the previous two series in which all grafts were surrounded by significant numbers of melanocytes. DISCUSSION
The results of this study leave no doubt that the mutations albino (c), steel (SZ), and viable dominant-spotting ( w’ ) produce their pigmentary defects through quite different pathways. Considerable evidence has accumulated in the past indicating that albinism is the result of the production of an abnormal enzyme tyrosinase, and that the melanocytes of c/c mice are incapable of producing pigment. Amelanotic melanocytes have been demonstrated repeatedly in the hair follicles of albino mice in the positions where melanocytes in normal mice are located (Chase et al., 1951; Silvers, 1956). These
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FIG. 4. Combination graft resulting from implantation of 13 clay Sld/SId skin with normal neural tube. All hairs are pigment-free. FIG. 5. Combination graft of Sld/SZdskin with normal neural tube. One pigmented hair is visible. The areasurroundingthe graft is pigment-free.
large clear cells are functionally similar to melanocytes in their sensitivity to X-irradiation (Quevedo, 1957), and skin from normal mice experimentally deprived of its neural crest lacks amelanotic melanocytes (Silvers, 1958). In the present study two additional tests were brought to bear on the action of the albino locus. First, albino skin which previously has received its own melanoblast complement is refractory to the invasion of melanoblasts when grafted with normal neural crest. It appears that the melanoblasts already present in the albino skin block the entrance of additional cells into the tissue. The influence of the c/c melanoblasts in preventing additional cells from entering the skin is quite rigid, for only 13% of the recovered grafts had just a few pigmented hairs present. The c/c skin had no influence on the migration of normal melanoblasts into the tissues of the host chick embryo and this observation was to be expected. Second, albino skin from ll-day embryos, prior to the time of its own melanoblast invasion, is receptive to normal melanoblast invasion and favors their differentiation. These results support the idea that the relationship between a given area of skin and its population of melanocytes is a rather constant one and appears to be established im-
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mediately after the melanoblasts initially migrate into an area. This ratio is set up well in advance of the establishment of a functional relationship between the melanocyte and the epidermal cells (epidermal melanin unit of Fitzpatrick and Breathnach, 1963). Very striking differences are evident when one compares the action of albinism to that of the dominant-spotting mutation. Skin from w‘ /WY embryos accepts melanoblasts from normal neural tubes and produces extensive hair growth with intense pigmentation. In this study, skin of ages 13-18 days was tested, and in a previous study 11-day skin was examined (Mayer and Green, 1968). The results were identical for all ages. It appears that melanoblasts never enter the skin of the WV/WV embryos since skin of all ages tested is equally receptive to normal melanoblasts. The critical age in this study is 13 days, for flank skin in embryos of this age should have received their melanoblast invasion just one day earlier. If melanoblasts do indeed enter the skin of W”lW’ embryos, then their presence should have been revealed by the grafting study. An alternative explanation proposed by Mintz (1969) is that WV/WV melanoblasts are preprogrammed for death at some later unspecified age. If their lethality is expressed soon after their arrival in the skin, then the results of this study and that of Mintz are in agreement. The fact that W’/+ heterozygotes possess a small amount of white-spotting could be explained on the basis of an early death of certain melanoblast clones, with a spreading in from adjacent areas of viable melanoblasts. Certainly the evidence from the present study is not consistent with the idea that undifferentiated or abnormally differentiated melanoblasts are present in WV/W’ skin at any age. As demonstrated by the albino series, if melanoblasts are present in the hair follicles, they are unmistakably revealed through blocking the entrance of other melanoblasts. No such action is apparent when dealing with WV/W’ skin. Although the action of the genes Sld and W’ mimic each other in the mouse, the behavior of SldlSld skin in the grafting series is closer to albino skin. Pigment formation is almost completely blocked in hair follicles when Sld/Sld skin is combined with normal neural tubes. In only a few grafts were pigmented hairs found, and their number was small. An interesting departure of the steel series from the albino, however, was the obvious lack of pigment in the host tissues surrounding Sld/Sld skin. The reason for this failure is difficult to explain, although two possibilities are immediately apparent.
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Steel skin may exhibit an inhibitory effect on melanoblast differentiation to the extent that pigment formation is blocked in the skin and in the host tissues. This inhibitor would have to be rather potent and diffusible in order to affect melanoblasts some distance from the skin location. Making the inhibitor of differentiation idea somewhat unlikely is the fact that in those few cases in which melanocytes were observed surrounding the graft, they were located immediately adjacent to the graft and not some distance away. The second possibility is that steel skin inhibits the migration of melanoblasts from the grafted neural tube. One can visualize how steel skin could prevent migration into itself, but the block to migration from neural tubes into the host tissues is more difficult to understand. Which ever of these two possible mechanisms proves to be correct, the action of SZd/Sld skin on the normal melanoblasts in this combination system is remarkably effective. This inhibitory action of steel skin is present throughout all the embryonic ages tested. Previous skin grafting studies using newborn or older mice have demonstrated that white-spotted skin has the capacity to support the differentiation of normal melanoblasts (Silvers and Russell, 1955). Steel skin was unfortunately not one of the mutants tested in these earlier studies. The present work shows that Sld is active throughout the embryonic ages 11-18 days, and perhaps even in adult mice. It is interesting to note, however, that hair follicles of all the white-spotting mutant mice examined were devoid of amelanotic melanocytes or cells recognizable as undifferentiated melanoblasts (Chase et al., 1951; Silvers 1956). These studies did include an examination of steel and dominant-spotting skin. There are few mutations in the mouse in which gene action through the skin environment affects the differentiation of melanoblasts. One notable example is agouti (Silvers and Russell, 1955). The environment of the hair follicle is the factor which determines whether the melanocytes will produce phaeomelanin or eumelanin. Melanocytes outside of the hair follicles in agouti mice are uniform in their production of eumelanin. Another mutation which appears to act specifically through the hair follicle is belted (Mayer and Maltby, 1964). Melanoblasts located in the white-spotted area are adversely affected and do not differentiate in or migrate into the hair follicles. The presence of melanoblasts in the white-spotted region of embryos 13 days of age can be demonstrated by grafting methods, and melanocytes were observed in the skin of the belt between the hair follicles of young mice. Additional evidence that bt
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acts specifically on the hair follicle comes from a study of melanocyte occurrence in tissues other than the skin. Normal and btlbt mice are identical in the occurrence of melanocytes in nonepidermal areas such as the harderian gland, leg musculature, and membranous labyrinth of the ear. In the case of steel, if the gene acts specifically through the skin in affecting melanoblast differentiation, one might similarly expect the other nonepidermal regions of the body to possessnormal pigmentation. This is not the case, and Sld/Sl” mice are devoid of pigmentation in all nonepidermal areas with the exception of the retinal epithelium. In this respect they are identical to W’IW” mice. The relationship between the pigment, blood, and germ cell defects in steel and dominant-spotting mice remains obscure. The anemia has been studied extensively in both mutants, and a comparison is now possible with the pigment system. Through the use of the spleen-colony assay method for hematopoietic stem cells it was found that the W-genes affect the number of stem cells or their capacity to form spleen colonies (McCulloch et al., 1964). The defect depends entirely on the genotype of the stem cells, and is completely independent of influences from other parts of the body. It can be recalled here that the pigment defect also resides in the stem melanoblasts and that the skin of WY/W’ mice has no adverse influence on their development. The evidence relating to pigment cells favors a reduced number or complete absence of melanoblasts, not a reduced capacity to form pigment. The stem hematopoietic cells of SlISl mice, on the other hand, are normal with respect to blood formation (McCulloch et al., 1965). Sl appears to exert its control by rendering the body incapable of supporting the proliferation or differentiation of hematopoietic cells to the extent that is possible in normal mice. Similarily, steel melanoblasts are normal in their ability to form pigment when presented with a favorable environment. The skin of Sld/Sld mice is incapable of supporting the proliferation, migration, or differentiation of the stem pigment cells. The evidence here favors the former two possibilities over the latter. Exactly how a single gene mutation might affect these two diverse cell types remains unclear. The sterility of both the W and Sl homozygotes results from a failure of the primordial germ cells to migrate from the yolk sac into the genital ridge between days 8 to 12 of fetal life. It appears that in both genotypes the primordial germ cells do not proliferate as rapidly as normal during this critical period, and this insufficiency may
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be responsible for their migratory failure (Bennett, 1956; Mintz, 1960). No experimental evidence is available to indicate whether this defect results from factors intrinsic to the primordial germ cells, or from an abnormality within the surrounding tissues. SUMMARY
The mutations albino, steel, and dominant-spotting were investigated through a method of introducing normal melanoblasts into skin from mutant embryos of various ages. The skin-melanoblast combinations were cultured in the chick embryo for 15 days, and the development of pigmentation in the grafted skin and host tissues was determined. Eleven-day embryonic albino skin combined with normal melanoblasts resulted in the appearance of pigment in the graft and host tissues. Thirteen day and older albino embryonic skinmelanoblast combinations yielded pigment only in the host tissues. Melanocytes were absent in the large majority of the grafts themselves. Thirteento 18-day WV/W’ embryonic skin-melanoblast combinations resulted in pigment formation in both the grafted skin and host tissues in all cases. SZd/SZd skin-melanoblast combinations of 13-18 days resulted in pigment development in neither the grafted skin nor the host tissues in the large majority of the cases. These results demonstrated that the presence of amelanotic melanoblasts in albino skin at 13 days and older prevented the entrance of normal melanoblasts. The receptivity of WV/W’ skin to normal melanoblasts indicated that skin of this genotype did not possess melanoblasts in any form at the ages tested. WV/WY melanoblasm either never entered the skin of prospective black-eyed white embryos, or they did not survive because of some intrinsic factor immediately after colonizing the skin. The skin of steel mice either prevented the differentiation of the normal melanoblasts or it inhibited their migration. This action of steel skin was rather potent, for the inhibition affected not only itself but the appearance of pigment in the host tissues as well. REFERENCES D. (1956). Developmental analysis of a mutation with pleiotropic effect in the mouse. J. Morphol. 98, 199-234. CHASE, H. B., F~AUCH, H., and SMITH, V. W. (1951). Critical stages of hair development and pigmentation in the mouse. Physiol. 2001. 24, l-10. COLEMAN, D. L. (1962). Effect of genie substitution on the incorporation of tyrosine into the melanin of mouse skin. Arch. Biochem. Biophys. 96, 562-568. BENNETT,
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FITZPATRICK, T. B., and BREATHNACH, A. S. (1963). Das epidermale Melanin-EinheitSystem. Dermatol. Wochenschr. 147, 481-489. LYON , M. F. (1968). Chromosomal and subchromosomal inactivation. Ann. Rev. Genet. 2, 31-52. MCCULLOCH, E. A., SIMINOVPTCH,L., and TILL, J. E. (1964). Spleen-colony formation in anemic mice of genotype WW’ Science 144, 844-846. MCCULLOCH, E. A., SIMINOVITCH, L., TILL, J. E., RUSSELL, E. S., and BERNSTEIN, S. E. (1965). The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype S1/Sld. Blood 26, 399-410. MAYER, T. C. (1965). The development of piebald spotting in mice. Develop. Biol. 11, 319-334. MAYER, T. C., and GREEN, M. C. (1968). An experimental analysis of the pigment defect caused by mutations at the Wand SI loci in mice. Deuelop. Biol. 18, 62-75. MAYER, T. C., and MALTBY, E. L. (1964). An experimental investigation of pattern development in lethal spotting and belted mouse embryos. Deuelop. Biol. 9, 269-286. MAYER, T. C., and REAMS, W. M., JR., (1962). An experimental analysis and description of the melanocytes in the leg musculature of the PET strain of mice. Anut. Rec. 142, 431-441. MINTZ, B. (1960). Embryological phases of mammalian gametogenesis. J. Cell. Camp. Physiol. 56, Suppl. 1, 31-47. MINTZ, B. (1967). Gene control of mammalian pigmentary differentiation. I. Clonal origin of melanocytes. Proc. Nut. Acad. Sci. U. S. 58, 344-351. MINTZ, B. (1969). Gene control of the mouse pigmentary system. Genetics 61, 41 (Abstr.). QUEVEDO, W. C., JR. (1957). Loss of clear cells in the hair follicles of X-irradiated albino mice. Amt. Rec. 127, 725-734. RAWLES, M. E. (1944). The migration of melanoblasts after hatching into pigment-free skin grafts of the common fowl. Physiol. 2001. 17, 167-183. RAWLES, M. E. (1947). Origin of pigment cells from the neural crest in the mouse embryo. Physiol. Zool. 20, 248266. RAWLES, M. E. (1955). Skin and its derivatives. In “Analysis of Development” (B. Willier, P. Weiss, and V. Hamburger, eds.), pp. 499-519. Saunders, Philadelphia. RUSSELL, E. S. (1954). Review of the pleiotropic effects of W-series genes on growth and differentiation. In “Aspects of Synthesis and Order in Growth” (Rudnick, D., ed.), pp. 113-126. Princeton Univ. Press, Princeton, New Jersey. SILVERS, W. K. (1956). Pigment cells: occurrence in hair follicles. J. Morphol. 99, 41-56. SILVERS, W. K. (1958). Origin and identity of clear cells found in hair bulbs of albino mice. Amt. Rec. 130, 135-144. SILVERS, W. K., and RUSSELL, E. S. (1955). An experimental approach to action of genes at the agouti locus in the mouse. J. Expl. Zool. 130, 199-220. WESTON, J. (1963). A radioautographic analysis of the migration and localization of trunk neural crest cells in the chick. Deuelop. Biol. 6, 279-310. WILLIER, B. H. (1952). Cells, feathers, and colors. Bios 23, 109-125.
BIOLOGY
23, 297-309 (1970)
A Comparison Albino,
of Pigment
Cell Development
in
Steel, and Dominant-Spotting Mutant
Mouse
Embryos1
THOMAS C. MAYER Department
of Biology,
Rider College, Trenton,
New Jersey 08602
Accepted July 16, 1970 INTRODUCTION
Melanin-synthesizing cells found in the coat of the mouse originate from the neural crest and migrate throughout the skin during days 8-12 of embryonic development (Rawles, 1947). Thirty-four primordial melanoblasts apparently are set aside early in the development of the mouse embryo (prior to day 7) and are lined up longitudinally, 17 on each side, during the initial formation of the neural crest (Mintz, 1967; however cf. Lyon, 1968). As revealed through studies on the chick embryo (Rawles, 1944; Weston, 1963), melanoblasts then undergo rapid proliferation and migrate laterally to occupy the skin up to a certain density. The wave of migrating cells continues to move into skin regions not occupied by melanoblasts, and migration ceases when all areas are occupied to their maximum density (Willier, 1952). The melanoblasts in the skin produce melanin long after the migration process is completed, and the sequence of their maturation does not correspond to the time of arrival of melanoblasts in a specific region (Rawles, 1955; Mayer and Reams, 1962). This complex process of early organization, migration, and differentiation of the melanoblast component of the neural crest is under genetic control, with the two obvious sites of control being the skin or the melanoblast itself. Disturbances in the organization of the primordial melanoblasts, their proliferation, migration, survival, or differentiation can result in defects or gaps in the coloration of the skin and hair. This report represents an analysis of the action of genes at three distinct genetic loci, albino (c), steel (Sl) and dominant-spotting (IV). ’ This investigation Foundation.
was supported
by Grant 297
GB 7989 from the National
Science
298
MAYER
Mice homozygous for mutant alleles at the W-locus are characterized by severe macrocytic anemia, lack of hair pigmentation and sterility (Russell, 1954). The pigmentation defect has been analyzed initially through the use of a grafting technique in which neural crest from normal or mutant mice was combined with skin from the appropriate genotype. The site of action of W appeared to be the neural crest. Skin of the mutant genotype did not influence normal melanoblasts in any adverse way (Mayer and Green, 1968). The mutant gene Sl is a very close mimic of W. Steel homozygotes are black-eyed white, anemic, and sterile (Bennett, 1956). This similarity, however, is a superficial one for the two genes appear to produce the pigment defect through different sites. Combination grafting has revealed that the skin of SlISl mice plays the determining role in the pigmentation block. The melanoblast component of the neural crest is normal in steel embryos (Mayer and Green, 1968). The action of the albino gene is apparently simpler and certainly better understood than the spotting genes. There are no obvious pleiotropic effects associated with c, and the pigmentation defect appears to be through the melanocyte at the level of the enzyme tyrosinase. Albino mice possess clear cells in the matrix of the hair follicles where melanocytes normally are located (Silvers, 1956). These clear cells are thought to be amelanotic melanocytes incapable of synthesizing melanin. In addition, tyrosinase activity is lacking in the skin of c/c mice, and is reduced in c/+ genotypes (Coleman, 1962). The results being presented in this report are a further characterization of the mechanism of action of the W, Sl, and c genes with respect to the pigmentation defect. The only previous experimental study on W and Sl mutant embryos utilized skin from 11-day embryos. Since at this age it was impossible to identify the genotype of the mutant embryos produced from matings of heterozygotes, the results were based on a small statistical sampling of the embryos produced. The differences revealed in this previous work between the action of Sl and W, and particularly the effect of SlISl skin on normal melanoblasts, made it essential that this work be repeated using embryos of known genotype. In addition to a confirmation of these previous results, it was anticipated that the use of whitespotted skin following the presumed migration of its own melanoblast population might yield information regarding the status of this population. A specific time of action of the steel inhibitory effect could also be assessed by using skin from embryos of progressively
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older ages. This study, therefore, was an extension of the previous report, and utilized skin from 13-day and older embryos, ages at which the genotype of the embryo could be accurately determined. The albino was included in this study in order to compare the behavior of skin with its own complement of amelanotic melanoblasts to that of white-spotted skin. MATERIALS
AND METHODS
Embryos used as skin donors in the W grafting series were produced from matings between C57BL/6J - W I+ parents. The W’IW embryos could be separated from their W’/+ and +I+ littermates by the pale color of the liver at ages 13 days and older. Steel skin donor embryos were from mating C57BL/6J-Sld/+ parents, and these homozygotes could likewise be identified by their pale livers at 13 days. Embryos of the A/J strain (ala b/b c/c) were used as skin donors for the albino series. In all cases the source of normal melanoblasts was 9 days neural tubes derived from embryos produced by mating C57BL/6J - +/+ parents. The combination grafting method has been described previously (Mayer, 1965). Briefly, the neural tubes from the posterior quarter of g-day embryos were removed and carefully cleaned of all adhering somite and skin cells with the aid of trypsin digestion. Skin from the midflank between the limb buds of 13-day or older embryos was removed with finely ground iridectomy scissors and cut into 1 mm square pieces which were suitable for grafting. One neural tube and one piece of skin were then grafted together to the flank of 2.5 day White Leghorn chick embryos. After a 15-day incubation period, the grafts were located, dehydrated, and cleared in oil of wintergreen. The presence or absence of pigment was then tabulated from the cleared whole mounts of the grafts without the use of stains or other treatment. The areas examined for pigment were the hair follicles of the graft and the skin and coelomic lining of the host surrounding the graft site. Each series of experiments included a group of skin grafts implanted without associated neural tubes. These control grafts served as a verification of the genotype of the W/IV and SldlSld donor embryos, and as a check for any host pigment formation resulting from the operating procedure itself. RESULTS
A total of 266 operations were performed from which 222 grafts were recovered for study. Thirty operated embryos failed to survive the incubation period, and 14 host embryos were recovered in which
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a graft was not found. Extensive hair growth was present in all the grafts tabulated. The results of the three mutant series are summarized in Table 1. Albino
Series
The results of this series were divided into two groups. In the older group skin was used from embryos 13-18 days of age. This skin had received its own complement or invasion of melanoblasts prior to grafting. In the younger group, 11-day skin prior to receiving its melanoblast invasion was used. In both groups combination grafts were made using normal g-day neural tubes. Twenty-three grafts were recovered from implantations using skin of the older group, and twenty of these produced hairs that were totally free of pigment. Hair growth in grafts was extensive with typically 30 to 40 hair follicles associated with each transplant. Another constant feature of these grafts was the extensive development of pigment in the tissues of the host chick embryo immediately surrounding the graft. Melanocytes were observed extending out in all directions from the graft for a distance of up to 3 mm. Although the extent of melanocyte migration into the host tissues varied somewhat from one graft to another, the pigmentation of the host was a characteristic and obvious feature of this series. Figure 1 is an example of a typical graft of this type. The remaining three grafts each possessed a few pigmented hairs, although the majority were pigTABLE SUMMARY
OF THE COMBINATION ASSOCIATION
With +/+
GRAFTING
WITH NORMAL
1 RESULTS USING MUTANT NEURAL
SKIN IN
TUBES”
Skin-only
tubes
Type
Hair w/w skin (13-18 days) SldlSld skin (13-18 days) c/c skin ( 13-18 days) c/c skin ( 11 days)
Coelom
Hair
Coelom
+cw
+(28)
-(21)
-(21)
- (60)
- (58) +(5)
-(31)
- (31)
+ -(3)
-cm
+(X9
-08)
-(18)
+-(3) +(22)
+cm
-U6)
-0.3
’ The skin-only column represents control grafts. + = pigment present; - = pigment absent; + and - = grafts having both pigmented and pigment-free hairs. Numbers represent grafts recovered of each type.
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FIG. 1. Combination graft resulting from the inplantation of 13-day c/c embryonic skin with normal neural tube. Note the pigment-free graft and hair follicles with a trail of melanocytes away from the graft into the host tissues.
ment-free. In these three cases, pigment cell migration into the host tissues was comparable to that from those grafts which completely lacked pigment in the hair follicles. It appeared likely that the cause of the pigmentation failure within the hair follicles of these older skin grafts was the presence of amelanotic melanoblasts already in the skin and occupying the spaces available to other melanoblasts. In order to clarify this point, a young skin series was undertaken in which 11-day albino skin was combined with normal (+ /+) neural tubes. Skin of this age had not yet received its own melanoblast invasion, and therefore did not possess a population of amelanotic melanoblasts. Twenty-two grafts were recovered, and all possessed extensive hair development with all hairs pigmented. In addition melanocytes were found within the host tissues surrounding the grafted skin. It appeared, therefore, that the presence of an amelanotic melanoblast population in the skin of the older series was an effective block to the entrance of additional normal melanoblasts. In conjunction with both the old and young grafting series, a group of skin-only control grafts was made. The purpose of the control series was to determine with certainty the origin of the melanocytes found in association with the combination grafts. Eighteen skin-only implantations of the older series and 16 of the younger series were recovered. Extensive hair growth was observed in the grafts of both series, but pigment was absent from both the graft and the host tissues in the operated area. These results clearly demonstrated that the operative procedure had no effect on the occurrence of pigment
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in the host, and that the melanocytes observed in the region of the graft were derived from the donor neural tubes. Dominant-Spotting
Series
Embryos presumed to be of the W’lW’ genotype because of the pale liver color were selected as skin donors. As an additional check of the donor genotype, skin-only grafts were made from each embryo used in the combination grafts. One embryo was discovered to have been incorrectly classified, and it was discarded from the tabulation of the results. The control grafts from all the other presumed W’/W’ embryos failed to produce pigment. Pigment-free hairs in large numbers were present in each graft, and the host tissues surrounding the operated area were devoid of melanocytes. Combination grafts in this series were produced by implanting skin from 13-18-day W’IW’ embryos in conjunction with g-day normal neural tubes. The results of these combination grafts were remarkably consistent. Twentyeight grafts were recovered, and all possessed large numbers of pigmented hairs (Fig. 2). In no case were any pigment-free hair follicles observed in grafts of this combination. The tissues of the host surrounding the graft were occupied by melanocytes in numbers essentially similar to that of the previous series (Fig. 3). Steel Series Presumed Sld/Sld embryos were identified in the same way as the WV/W’ embryos, and skin-only grafts were made to verify the genotype. One embryo was incorrectly classified, and the error was apparent from the control series. Thirty-one skin-only grafts were recovered from Sld/Sld skin implantations, and all were devoid of pigment both in the graft and in the surrounding host tissues. Sixtythree grafts were recovered in this series from combining skin 13 to 18 days of age with g-day normal neural tubes. The large majority of these grafts produced only pigment-free hairs (Fig. 4). Hair growth from the SldlSld skin was extensive and the relationship of the grafted skin to the neural tube at the time of recovery was identical to the previous two series. The absence of pigment from the steel hair follicles was not absolute, for in a few grafts some pigmented hairs were found along with those lacking pigment (Fig. 5). Only three cases of these mixed grafts were found from the total of 63 recovered cases. The occurrence of melanocytes in the host tissues was also of particular interest in this series. In only five cases were
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FIG. 2. Combination graft of 13 day W‘/W’skin with normal neural tube. Intense pigmentation of hair follicles is obvious. FIG. 3. Combination graft of 13 day W /W skin with normal neural tube. Migration of melanocytes into host tissue is extensive.
melanocytes found in the host tissues surrounding the graft. The area of the host immediately adjacent to the remaining 58 grafts of this series was completely devoid of melanocytes. This finding is particularly interesting when compared to the previous two series in which all grafts were surrounded by significant numbers of melanocytes. DISCUSSION
The results of this study leave no doubt that the mutations albino (c), steel (SZ), and viable dominant-spotting ( w’ ) produce their pigmentary defects through quite different pathways. Considerable evidence has accumulated in the past indicating that albinism is the result of the production of an abnormal enzyme tyrosinase, and that the melanocytes of c/c mice are incapable of producing pigment. Amelanotic melanocytes have been demonstrated repeatedly in the hair follicles of albino mice in the positions where melanocytes in normal mice are located (Chase et al., 1951; Silvers, 1956). These
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FIG. 4. Combination graft resulting from implantation of 13 clay Sld/SId skin with normal neural tube. All hairs are pigment-free. FIG. 5. Combination graft of Sld/SZdskin with normal neural tube. One pigmented hair is visible. The areasurroundingthe graft is pigment-free.
large clear cells are functionally similar to melanocytes in their sensitivity to X-irradiation (Quevedo, 1957), and skin from normal mice experimentally deprived of its neural crest lacks amelanotic melanocytes (Silvers, 1958). In the present study two additional tests were brought to bear on the action of the albino locus. First, albino skin which previously has received its own melanoblast complement is refractory to the invasion of melanoblasts when grafted with normal neural crest. It appears that the melanoblasts already present in the albino skin block the entrance of additional cells into the tissue. The influence of the c/c melanoblasts in preventing additional cells from entering the skin is quite rigid, for only 13% of the recovered grafts had just a few pigmented hairs present. The c/c skin had no influence on the migration of normal melanoblasts into the tissues of the host chick embryo and this observation was to be expected. Second, albino skin from ll-day embryos, prior to the time of its own melanoblast invasion, is receptive to normal melanoblast invasion and favors their differentiation. These results support the idea that the relationship between a given area of skin and its population of melanocytes is a rather constant one and appears to be established im-
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mediately after the melanoblasts initially migrate into an area. This ratio is set up well in advance of the establishment of a functional relationship between the melanocyte and the epidermal cells (epidermal melanin unit of Fitzpatrick and Breathnach, 1963). Very striking differences are evident when one compares the action of albinism to that of the dominant-spotting mutation. Skin from w‘ /WY embryos accepts melanoblasts from normal neural tubes and produces extensive hair growth with intense pigmentation. In this study, skin of ages 13-18 days was tested, and in a previous study 11-day skin was examined (Mayer and Green, 1968). The results were identical for all ages. It appears that melanoblasts never enter the skin of the WV/WV embryos since skin of all ages tested is equally receptive to normal melanoblasts. The critical age in this study is 13 days, for flank skin in embryos of this age should have received their melanoblast invasion just one day earlier. If melanoblasts do indeed enter the skin of W”lW’ embryos, then their presence should have been revealed by the grafting study. An alternative explanation proposed by Mintz (1969) is that WV/WV melanoblasts are preprogrammed for death at some later unspecified age. If their lethality is expressed soon after their arrival in the skin, then the results of this study and that of Mintz are in agreement. The fact that W’/+ heterozygotes possess a small amount of white-spotting could be explained on the basis of an early death of certain melanoblast clones, with a spreading in from adjacent areas of viable melanoblasts. Certainly the evidence from the present study is not consistent with the idea that undifferentiated or abnormally differentiated melanoblasts are present in WV/W’ skin at any age. As demonstrated by the albino series, if melanoblasts are present in the hair follicles, they are unmistakably revealed through blocking the entrance of other melanoblasts. No such action is apparent when dealing with WV/W’ skin. Although the action of the genes Sld and W’ mimic each other in the mouse, the behavior of SldlSld skin in the grafting series is closer to albino skin. Pigment formation is almost completely blocked in hair follicles when Sld/Sld skin is combined with normal neural tubes. In only a few grafts were pigmented hairs found, and their number was small. An interesting departure of the steel series from the albino, however, was the obvious lack of pigment in the host tissues surrounding Sld/Sld skin. The reason for this failure is difficult to explain, although two possibilities are immediately apparent.
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Steel skin may exhibit an inhibitory effect on melanoblast differentiation to the extent that pigment formation is blocked in the skin and in the host tissues. This inhibitor would have to be rather potent and diffusible in order to affect melanoblasts some distance from the skin location. Making the inhibitor of differentiation idea somewhat unlikely is the fact that in those few cases in which melanocytes were observed surrounding the graft, they were located immediately adjacent to the graft and not some distance away. The second possibility is that steel skin inhibits the migration of melanoblasts from the grafted neural tube. One can visualize how steel skin could prevent migration into itself, but the block to migration from neural tubes into the host tissues is more difficult to understand. Which ever of these two possible mechanisms proves to be correct, the action of SZd/Sld skin on the normal melanoblasts in this combination system is remarkably effective. This inhibitory action of steel skin is present throughout all the embryonic ages tested. Previous skin grafting studies using newborn or older mice have demonstrated that white-spotted skin has the capacity to support the differentiation of normal melanoblasts (Silvers and Russell, 1955). Steel skin was unfortunately not one of the mutants tested in these earlier studies. The present work shows that Sld is active throughout the embryonic ages 11-18 days, and perhaps even in adult mice. It is interesting to note, however, that hair follicles of all the white-spotting mutant mice examined were devoid of amelanotic melanocytes or cells recognizable as undifferentiated melanoblasts (Chase et al., 1951; Silvers 1956). These studies did include an examination of steel and dominant-spotting skin. There are few mutations in the mouse in which gene action through the skin environment affects the differentiation of melanoblasts. One notable example is agouti (Silvers and Russell, 1955). The environment of the hair follicle is the factor which determines whether the melanocytes will produce phaeomelanin or eumelanin. Melanocytes outside of the hair follicles in agouti mice are uniform in their production of eumelanin. Another mutation which appears to act specifically through the hair follicle is belted (Mayer and Maltby, 1964). Melanoblasts located in the white-spotted area are adversely affected and do not differentiate in or migrate into the hair follicles. The presence of melanoblasts in the white-spotted region of embryos 13 days of age can be demonstrated by grafting methods, and melanocytes were observed in the skin of the belt between the hair follicles of young mice. Additional evidence that bt
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acts specifically on the hair follicle comes from a study of melanocyte occurrence in tissues other than the skin. Normal and btlbt mice are identical in the occurrence of melanocytes in nonepidermal areas such as the harderian gland, leg musculature, and membranous labyrinth of the ear. In the case of steel, if the gene acts specifically through the skin in affecting melanoblast differentiation, one might similarly expect the other nonepidermal regions of the body to possessnormal pigmentation. This is not the case, and Sld/Sl” mice are devoid of pigmentation in all nonepidermal areas with the exception of the retinal epithelium. In this respect they are identical to W’IW” mice. The relationship between the pigment, blood, and germ cell defects in steel and dominant-spotting mice remains obscure. The anemia has been studied extensively in both mutants, and a comparison is now possible with the pigment system. Through the use of the spleen-colony assay method for hematopoietic stem cells it was found that the W-genes affect the number of stem cells or their capacity to form spleen colonies (McCulloch et al., 1964). The defect depends entirely on the genotype of the stem cells, and is completely independent of influences from other parts of the body. It can be recalled here that the pigment defect also resides in the stem melanoblasts and that the skin of WY/W’ mice has no adverse influence on their development. The evidence relating to pigment cells favors a reduced number or complete absence of melanoblasts, not a reduced capacity to form pigment. The stem hematopoietic cells of SlISl mice, on the other hand, are normal with respect to blood formation (McCulloch et al., 1965). Sl appears to exert its control by rendering the body incapable of supporting the proliferation or differentiation of hematopoietic cells to the extent that is possible in normal mice. Similarily, steel melanoblasts are normal in their ability to form pigment when presented with a favorable environment. The skin of Sld/Sld mice is incapable of supporting the proliferation, migration, or differentiation of the stem pigment cells. The evidence here favors the former two possibilities over the latter. Exactly how a single gene mutation might affect these two diverse cell types remains unclear. The sterility of both the W and Sl homozygotes results from a failure of the primordial germ cells to migrate from the yolk sac into the genital ridge between days 8 to 12 of fetal life. It appears that in both genotypes the primordial germ cells do not proliferate as rapidly as normal during this critical period, and this insufficiency may
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be responsible for their migratory failure (Bennett, 1956; Mintz, 1960). No experimental evidence is available to indicate whether this defect results from factors intrinsic to the primordial germ cells, or from an abnormality within the surrounding tissues. SUMMARY
The mutations albino, steel, and dominant-spotting were investigated through a method of introducing normal melanoblasts into skin from mutant embryos of various ages. The skin-melanoblast combinations were cultured in the chick embryo for 15 days, and the development of pigmentation in the grafted skin and host tissues was determined. Eleven-day embryonic albino skin combined with normal melanoblasts resulted in the appearance of pigment in the graft and host tissues. Thirteen day and older albino embryonic skinmelanoblast combinations yielded pigment only in the host tissues. Melanocytes were absent in the large majority of the grafts themselves. Thirteento 18-day WV/W’ embryonic skin-melanoblast combinations resulted in pigment formation in both the grafted skin and host tissues in all cases. SZd/SZd skin-melanoblast combinations of 13-18 days resulted in pigment development in neither the grafted skin nor the host tissues in the large majority of the cases. These results demonstrated that the presence of amelanotic melanoblasts in albino skin at 13 days and older prevented the entrance of normal melanoblasts. The receptivity of WV/W’ skin to normal melanoblasts indicated that skin of this genotype did not possess melanoblasts in any form at the ages tested. WV/WY melanoblasm either never entered the skin of prospective black-eyed white embryos, or they did not survive because of some intrinsic factor immediately after colonizing the skin. The skin of steel mice either prevented the differentiation of the normal melanoblasts or it inhibited their migration. This action of steel skin was rather potent, for the inhibition affected not only itself but the appearance of pigment in the host tissues as well. REFERENCES D. (1956). Developmental analysis of a mutation with pleiotropic effect in the mouse. J. Morphol. 98, 199-234. CHASE, H. B., F~AUCH, H., and SMITH, V. W. (1951). Critical stages of hair development and pigmentation in the mouse. Physiol. 2001. 24, l-10. COLEMAN, D. L. (1962). Effect of genie substitution on the incorporation of tyrosine into the melanin of mouse skin. Arch. Biochem. Biophys. 96, 562-568. BENNETT,
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FITZPATRICK, T. B., and BREATHNACH, A. S. (1963). Das epidermale Melanin-EinheitSystem. Dermatol. Wochenschr. 147, 481-489. LYON , M. F. (1968). Chromosomal and subchromosomal inactivation. Ann. Rev. Genet. 2, 31-52. MCCULLOCH, E. A., SIMINOVPTCH,L., and TILL, J. E. (1964). Spleen-colony formation in anemic mice of genotype WW’ Science 144, 844-846. MCCULLOCH, E. A., SIMINOVITCH, L., TILL, J. E., RUSSELL, E. S., and BERNSTEIN, S. E. (1965). The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype S1/Sld. Blood 26, 399-410. MAYER, T. C. (1965). The development of piebald spotting in mice. Develop. Biol. 11, 319-334. MAYER, T. C., and GREEN, M. C. (1968). An experimental analysis of the pigment defect caused by mutations at the Wand SI loci in mice. Deuelop. Biol. 18, 62-75. MAYER, T. C., and MALTBY, E. L. (1964). An experimental investigation of pattern development in lethal spotting and belted mouse embryos. Deuelop. Biol. 9, 269-286. MAYER, T. C., and REAMS, W. M., JR., (1962). An experimental analysis and description of the melanocytes in the leg musculature of the PET strain of mice. Anut. Rec. 142, 431-441. MINTZ, B. (1960). Embryological phases of mammalian gametogenesis. J. Cell. Camp. Physiol. 56, Suppl. 1, 31-47. MINTZ, B. (1967). Gene control of mammalian pigmentary differentiation. I. Clonal origin of melanocytes. Proc. Nut. Acad. Sci. U. S. 58, 344-351. MINTZ, B. (1969). Gene control of the mouse pigmentary system. Genetics 61, 41 (Abstr.). QUEVEDO, W. C., JR. (1957). Loss of clear cells in the hair follicles of X-irradiated albino mice. Amt. Rec. 127, 725-734. RAWLES, M. E. (1944). The migration of melanoblasts after hatching into pigment-free skin grafts of the common fowl. Physiol. 2001. 17, 167-183. RAWLES, M. E. (1947). Origin of pigment cells from the neural crest in the mouse embryo. Physiol. Zool. 20, 248266. RAWLES, M. E. (1955). Skin and its derivatives. In “Analysis of Development” (B. Willier, P. Weiss, and V. Hamburger, eds.), pp. 499-519. Saunders, Philadelphia. RUSSELL, E. S. (1954). Review of the pleiotropic effects of W-series genes on growth and differentiation. In “Aspects of Synthesis and Order in Growth” (Rudnick, D., ed.), pp. 113-126. Princeton Univ. Press, Princeton, New Jersey. SILVERS, W. K. (1956). Pigment cells: occurrence in hair follicles. J. Morphol. 99, 41-56. SILVERS, W. K. (1958). Origin and identity of clear cells found in hair bulbs of albino mice. Amt. Rec. 130, 135-144. SILVERS, W. K., and RUSSELL, E. S. (1955). An experimental approach to action of genes at the agouti locus in the mouse. J. Expl. Zool. 130, 199-220. WESTON, J. (1963). A radioautographic analysis of the migration and localization of trunk neural crest cells in the chick. Deuelop. Biol. 6, 279-310. WILLIER, B. H. (1952). Cells, feathers, and colors. Bios 23, 109-125.