Genetic effects on fine structure and development of pigment granules in mouse hair bulb melanocytes

DEVELOPMEiiTAL

Genetic Pigment

17, 351-365

BIOLOGY

(1968)

Effects on Fine Structure Granules

in Mouse I. The b and

ELIZABETH Department

of Zoology,

University Accepted

and Development

of

Hair Bulb Melanocytes d Loci’

RITTENHOUSE’ of

ivfichigan, Ann Arbor, Michigan

September

48104

22, 1967

INTRODUCTION

The formation of mammalian melanin pigment granules involves the formation of a framework, thought to be protein and to have tyrosinase activity (Seiji et al., 1963) upon which a layer of electron dense melanin appears and thickens until the original structure of the granule is obscured. The framework, which at some stage of granule formation becomes wholly or partly surrounded by an external membrane, may appear as a bundle of parallel rods or fibers (Moyer. 1963; Rappaport et al., 1963; Wellings and Siegel, 1963), a rolled membrane (Birbeck and Barnicot, 1959; Dowling and Gibbons, 1962; Wellings and Siegel, 1963; Moyer, 1966), a three-dimensional crystal lattice (Drochmans, 1960, 1963)) or an aggregation of round particles (Wellings and Siegel, 1959). It has been described as forming within enlarging Golgi vesicles in the human hair bulb melanocyte (Birbeck and Barnicot, 1959) and as arising by aggregation of strands connected with free ribosomes in mouse retinal pigment epithelium (Moyer, 1963). The studies of human hair bulb melanocytes and mouse retinal melanocytes show that granule structure can vary with color genotype ‘Part of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Urriversity of Michigan. This work was (subsequently GMsupported in part by USPHS Genetics traineeship 2G-71-C3 7107)) USPHS research grant CA-04305 (subsequently NIH grant HD-01254 ), and NIH training grant 2G-989/62-3. ‘Present address: Department of Genetics, Albert Einstein College of Meditine, New York, New York 10461. 351 @ 1968 by Academic

Press Inc.

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and suggest that formation and structure of granules may differ in different species or in melanocytes of different location and embryonic origin. This paper will deal with the structure and formation of black and brown granules in hair bulb melanocytes of the house mouse, and also with the effect of mutation at the Maltese dilution (d) locus. Light microscopy (Russell, 1949) indicates that the d allele produces a pale coat color chiefly by altering the distribution of pigment granules, rather than the quantity of pigment, in the hair shaft, and especially by causing a large percentage of the granules to be arranged in clumps. MATERIALS

AND METHODS

Dorsal skin from S-day-old mice was fixed in Veronal-acetate buffered osmium tetroxide solution, embedded in Epon according to the procedure of Luft (1961) or in Maraglas according to the procedure of Freeman and Spurlock ( 1962), and sectioned on a PorterBlum microtome. Sections were stained in an aqueous solution of uranyl acetate and examined in an RCA EMU 3E microscope. All mice came from stocks maintained at the Mammalian Genetics Center of the University of Michigan. All were non-agouti (au), pigmented (CC), and free of dilution factors other than Maltese. For each color genotype, skin from at least three mice, no two having the same parents, was examined. The intense black mice (BBDD) were C57BL/6J. The Maltese black (BBdd) mice came from a stock Two intense obtained by crossing C57BL/6J- at with STOLI/Lw. brown (bbDD) mice were bb offspring of YBR/HeWiHa matings, and the third came from a stock obtained by crossing C57Br/cd with Cordovan. The Maltese brown mice were DBAIBJ. RESULTS

The Melunocyte The melanocyte in the hair bulb of the house mouse is a dendritic cell with an extensive granular endoplasmic reticulum (Fig. 1) and an extensive Golgi region (Figs. 2 and 7) consisting of many smoothwalled vesicles and a few stacks of smooth double membranes. In many sections the endoplasmic reticulum and the Golgi membranes lie chiefly on opposite sides of the nucleus. Around the periphery of the Golgi region, next to the cell membrane or nuclear membrane, is

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a region containing many free ribosomes and a few granular membranes (Fig. 4). Extensions of this granular region invade the Golgi region (Figs. 2 and 7). Mitochondria are found throughout the cytoplasm but are infrequent in the Golgi region. Some sections also contain bodies consisting of an external membrane enclosing a group of vesicles resembling Golgi vesicles (Fig. 8). Such bodies may contain, besides vesicles, a circular area of uniform density slightly less than the density of melanin. These vesicle-containing bodies have the general appearance of multivesicular bodies or lysosomes. Melanin granules are found throughout the cytoplasm, but lightly melanized and unmelanized granules are most common toward the edge of the Golgi region, where free ribosomes, rough endoplasmic retieulum, and smooth-walled vesicles occur together (Figs. 2 and 4).

ln,tense black (BBDD) and Malt,ese black (BBdd). Sections of black melanocytes (Figs. l-7) contain a mixture of round and elongate melanized bodies suggesting a population of granules round in cross section and oval in longitudinal section. In areas o&side the Golgi region (Figs. 1 and 7) most granules consist of a uniformly dense melanin deposit, with a persisting external membrane sometimes visible (Fig. 1). They may be as long as 1 p. Granules in the Golgi region are smaller and often show internal structure. In presumed longitudinal sections the pattern is one of parallel bars of melanization (Figs. 2 and 4). In cross section the pattern is more variable ( Figs. 24). Sometimes it is a spiral, or a pair of complete or incomplete concentric circles, but often it is much less regular. Most of the longitudinal sections show two or three bars, but a few show as many as seven (Fig. 7). The fact that the presumed cross sections very rarely suggest a structure that would show more than four bars in longitudinal section suggests that the granules with six or sevrn bars should be considered as unusual granules, not as evidence that most granules would show more than four bars if cut exactly through the center. Unmelanized and very lightly melanized granules in longitudinal section show a regular striation along the longitudinal strands, and the first visible melanin appears on the dense lines of this striation (Fig. 7). Very rarely, an unmelanized bodv consists of a single faintly striated area \yithin the external membrane (Fig. 5).

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FIG. 1. BBDD. Region of the endoplasmic reticulum of a melanocyte, with large, uniformly dense granules. The forked mass of melanin on the right act ually consists of two distinct granules which cannot be resolved on the print. The

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The appearance of granules in various stages of melanization indicates that the internal framework on which melanin is deposited consists, in its simplest form, of a single membrane wrapped in jelly-roll fashion. Many granules, however, apparently contain strips or tubes of membrane in some other arrangement. These differences arc’ evident chiefly in cross section, the appearance of parallel strands in longitudinal section being little disturbed. Umnelanized bodies like the one in Fig. 5 could be interpreted as unmei.anized granules cut so that a large region of internal membrane lies in the plane at section.

FIG. 3. Patterns seen in presumed melanized black granules.

cross sections of lightly

melanized

and un-

It is possible that large unmelanized or lightly melanized granules showing more than four strands in longitudinal section are more common in Maltese black melanocytes than in intense black melanocytes, but in general the granules in the Golgi regions of the two genotypes are similar in size, number, and structure. Outside the Golgi region the individual granules are similar, but the Maltese black melanocyte (Fig. 7) is much more densely crowded with granules. There is nothing in the appearance of the granules themselves to indicate the cause of this crowding. There is no appearance of separate clumps within the cell, but only of the presence of a very large number of granules closely packed throughout all the cytoplasm outside the Golgi region. A similar spacing of granules is seen in the clumps within Maltese hair cells (Fig. 12). Intense brown (bbDD) and Maltese brown (bbdd). Sections through intense brown and Maltese brown melanocytes suggest that intense brown granules are usually nearlv spherical, while Maltese left-hand granule of thi5 pair is slight11 more than 1 p long. Arrows indicate faintl! visible external membrane. Maraglas embedded. x 30,970. FK:. 2. RBDD. Golgi region of a melanocyte. Some rough-surfaced membranes (clr) lie in the area of Golgi membranes. Granules g, and e are longitudinal sections with the structure of longitudinal bars faintly distinguishable. Cranlde g: is a cross section showing a spiral pattern of melanization. The arrow indicates a body consisting of a membrane-enclosed clump of small vesicles. The enclosed area resembles the area below and to the lvft of thr botlx. Epon ~IIIhedd~vl. x 30.970.

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brown granules are usually oval in longitudinal section and round in cross section. In sections of unmelanized granules of both genotypes the framework appears as a disorderly tangle of fine filaments (Figs. S-10). In some cases the framework shows many small circles wbieb might be interpreted as very fine-walled vesicles or as loops and rings of strands (Fig. 10). The structure of unmelanized intense and Maltese brown granules appears, in general, to be a ball of looseI> tangled strands, but might conceivably be a spongelike system of narrow membranes. Sometimes one end of an oval granule is unmelanized while the other has a densely melanized area resembling a rosette ( Figs. 10 and 11). Such a pattern does not resemble the cross section of a rolled membrane in a black granule, however, having more the appearance of an alignment of small strands and circles. It occurs in oval granules of either genotype, but is more common in Maltese brown granules, which occasionally have two or even three centers of melanization (Fig. 11). This difference may result entirel) from the greater proportion of oval granules in Maltese brown melanocytes. In granules of all types and all degrees of melanization there is usually some unmelanized or lightlv melanizecl framework just inside the external membrane. Even in large granules the melanin deposit rarely has the uniform densitv characteristic of black melanin deposits ( Fig. 9 ) . FK:. 4. BBDD. The cell membrane, with adjacent ribosomal region, runs ;*cross the top of the picture. The stacks of Golgi membranes are distended in several places, but the swollen area shows no structure suggesting granule formation. Granule gl, apparently a cross section, shows a more complex structure than a simple spiral. In the lower right-hand corner of the picture one arrow indicates a smooth-surfaced bulge at the end of a rough-surfaced membrane pair and the other indicates two smooth-walled vesicles similar to the bulge but showing faint internal structure. The internal structure of the lower vesicle, especially, suggests the formation of granule framework. g? is a longitudinal section of a granule, with obvious parallel bars still showing a faint pcriodicity. Clusters of rihcscmcs lie near both ends of this granule. No membrane ti visible around the unmelanixed extentions of the upper ends of the longitudinal bars. Epcn embed&d. x 44,000. FIG 5. BBDD.

The area of ill-defined striation suggests a membrane lying in the plane of section. At the upper tip of the granule the external membrane appears to be folded back, leaving an opening to the surrounding cytoplasm. Epon embedded. x 61,000. FIG. 6. BBDD. The parallel strands at the upper tip of the granule seem to extend, as darker lines, into a region of ,ribosomes, Epon embedded. X61,000.

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FIG. 7. BBdd. The crowding of granules characteristic of this genotype can be aecn at the upper right-hand corner of the picture. Granule gl (enlarged to

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In most cells the large, heavily melanized granules (up to 0.75 P greatest diameter in intense brown and 0.8~ in Maltese brown) are distinctly larger than the largest unmelanized granules (up to 0.55 ,U greatest diameter). Periodicity of the type seen in longitudinal strands of black granules is not obvious in brown granules. Possibly it is not truly absent, but simply difficult to identify in granule framework showing very few long lines and almost no parallel lines. The grain of the embedding medium can produce an effect of very fine periodicity in any membrane, and an alignment of short strands or vesicles can suggest a coarser periodicity. Many brown granules have some appearance of periodicity, but it cannot be definitely identified as corresponding to the periodicity readily seen in black granules. Granule formation and pwth. Melanin granules classified as immature on the basis of their small size and visible internal structure are most common at the periphery of the Golgi region where smoothwalled vesicles, free ribosomes, and occasional rough-surfaced membranes of granular endoplasmic reticulum occur together. The strands of brown granule framework are often associated with small dense points resembling the free ribosomes seen nearby (Fig. S), but it is possible that these small particles within the granules represent the first appearance of melanin. Particles resembling ribosomes were not seen within black granules, but some sections suggest a relationship between unmelanized granule framework and ribosomes lying just outside the granule. In Fig. 4, unmelanized framework of granule g? seems to extend beyond the external membrane into a region of ribosomes. In Fig. 6 continuations of the parallel strands extend as ~40,000 in the inset) shows obvious striation along its longitudinal strands. The arrow indicates a region in which the striation is emphasized by small dens< points which may indicate the beginning of melanization. A few rough-surfaced membranes (er) appear in the Golgi area. Epon embedded. ~27,800. Fm. 8. bbDD. A lysosomelike body (arrow), with a dense, structureless inclusion, lies near several bodies which resemble it except for the absence of the dense inclusion. gI is an unmelanized or very lightly melanized granule with a numbr~r of small, dense regions resembling the ribosomes in the surrounding cvtop 1asm. Epon embedded. x 34,000. FIG 9. bbDD. Peripheral region of one melanocyte and dendrite (D) of another. The granules of the dendrite are distinctly larger than the granules of the peripheral cell body. All granules show, in some degree, the rather looseI\ packed peripheral region and irregular melanin surface characteristic of granules seen at the upper right-hand comer of the picture. Granule g, (enlarged to

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Fm. 10. bbdd. The large number of granules in this rather small area is characteristic of this genotype. One unmelanized granule and three lightly

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dense lines into a region of ribosomes. Such formations give the impression of a granule framework being “spun out” from ribosomcs at one end of the granule. Although the small Golgi vesicles are strongly implicated in granule formation both by their abundance and by their proximity to the immature granules, there is little evidence about the nature of their contribution. In the brown genotypes there is little to suggest that granules originate by enlargement of a single vesicle. In sections of the black melanocytes such an interpretation is sometimes possible (Fig. 4), but is complicated by the similar size of the larger Golgi vesicles and cross sections of immature granules. Melanocytes of all four genotypes occasionally contain bodies consisting of an area not obviously different from the surrounding region of smooth-walled vesicles, but wholly or partly cut off from it by a membrane or by an alignment of vesicles or double membranes (Figs. 2 and 11). These bodies resemble immature granules in size and location, and differ somewhat from the multivesicular or lysosomelike bodies, which have a paler appearance caused by wider spacing of internal vesicles and tend to have a more irregular outline. A lack of convincing transitional stages, however, suggests that these bodies do not represent the origin of melanin granules, but are more likelv to represent the origin of multivesicular bodies. DISCUSSION

The pattern of pigment granule framework in hair bulb melanocytes of the housr mouse is greatlv influenced bv the h locus and oul\, melanized granules form a row across the top of the picture. The two middle granules in this row show the off-center pattern of melanization often seen in this genot)-pe. ‘\luch of the structure of these granules seems to consist of small circles, possible representing fine-walled vesicles. The melanized patterns ol granules .g, and g: seem to involve alignment and coalescence of small circles. loops, and strands. Epon embedded. ~44,000. FIG. 11. bbcld. Granule gl has a framework organized as two distinct centers. The Inore heavily melanized center appears to consist of a cluster of melanizing circles. The outline of granule gj suggests three centers of melanization. Thrv arrow indicates a membrane enclosing several small vesicles of the Golgi type. Two large membrane-enclosed bodies containing similar, but widely separated, vesicles are seen at the right. Epon embedded. ~33,000. FIG. 12. bbdd. Clump of granules in a hair cell. This clump is unusual in that it appears to be partly surrounded by a membrane which is probably thr cell membrane of either the melanocyte or the hair cell. Epon embedded. x 22,000.

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influenced by the d locus. The framework of BBDD and BBdd granules, although not often quite as simple as a single rolled membrane, usually involves a very few membranes, each extending almost the full length of the granule. Unmelanized bbDD and bbdd granules, on the other hand, resemble loosely tangled balls of filaments or spongelike aggregations of small strips and pieces of membrane. Moyer (1963, 1966) has reported that in mouse retina the b allele affects the appearance of the deposited melanin, brown melanin being more coarsely granular, but does not cause any disruption of the pattern of granule framework. In brown hair bulb granules the disorderly framework itself produces melanin deposits of coarsely granular appearance and irregular surface, and it is difficult to judge whether any of this appearance arises from properties of the melanin itself. Both melanized and unmelanized granules of the Golgi region are smaller than the melanized granules outside the Golgi region, but it is not clear whether this enlargment involves addition of new framework at the periphery of the granule or results entirely from thickening of the deposit of melanin on the original framework. The appearance of the black granules provides little support for either possibility. Melanized framework occurs with definitely identifiable unmelanized framework only in the small, lightly melanized granules, On the other hand, it appears that in the nearly mature granules the substrate would have to penetrate a very thick layer of polymerized melanin to reach a melanin-forming enzyme system located on the original framework. The fact that all but the smallest granules are uniformly dense prevents direct observation of any thickening of melanin on individual membrane layers, and the poor visibility immediately surrounding a large melanin deposit, which often makes it difficult to determine whether the external membrane is still present, might also obscure the presence of new framework closely applied to the melanized region. The appearance of the brown granules is more favorable to both possibilities. Since very few granules have uniformly dense centers it is easier to accept the penetration of substrate as possible. Most granules also show some lightly melanized or unmelanized framework just inside the external membrane, and this material might be interpreted as newly added framework. Granules of the type shown in Fig. 5 have been interpreted as membranes lying in the plane of section, rather than as sections slightly

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through three-dimensional lattices such as Drochmans ( 1960, 1963) has described in human condyloma, because of the absence of bodies interpretable as cross sections of three-dimensional lattices. The granule of Fig. 6, however, with its pale matrix and thick strands of lowcontrast periodicity merging into a striated area, differs from the other immature granules seen and resembles the pictures interpreted by Drochmans as helical strands coalescing to produce the three-dimensional lattice. Since only one other granule with similar strands and matrix (and without the striated area) was seen in the study of these four genotypes and of seven others (Rittenhouse. 1968 ), they must be regarded as atypical in this material. Although Maltese dilution has some effect on granule shape and size, especially in bb melanocytes, the characteristic crowding of dd granules in the melanocyte is not attributable to any visible property of granule structure or formation. In the most denselvi crowded areas there may be contact between the membranes of adjacent granules. but there is nothing to suggest that this is the cause rather than the effect of the crowding. In the less crowded Golgi region such contact is no more frequent in dd than in DD melanocytes. There is no other indication of binding or fusion of granules to one another. Moyer (1966) has described dd granules in methacrylate-embedded melanocvtes as being bound to one another and to the nuclear membrane bfr coarse strands, but the coarsely precipitated appearance of the cytoplasm in both DD and dd methacrvlate-embedded material creates difficulties of interpretation. It has been shown by Straile (1964) that granules may be released into the hair cells from the cell body as well as from the tips of the dendrites. dd melanocytes have fewer and thinner dendrites than DD melanocytes (Markert and Silvers, 1956). In the absence of any other visible basis for the formation of granule clumps, it seems likely that inadequate development of the dendritic system leads to accumulation of granules in the cell body, and that release of these crowded granules from the cell bodv produces granule clumps in the hair cells. SUhISIARY

This study dealt with the effect of mutation at the 1) and cl loci on the fine structure of melanin granules in the hair bulb melanocytes of the house mouse. Mutation at the b locus affects both shape and structure of the granules. BB granules are oval, and the structure of

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the unmelanized granules consists of one or more rolled membranes. bb granules are round, and their internal framework resembles a tangled ball of strands. The principal effect of the d a11e1e on the melanocyte is an increase in the number of granules present, apparently resulting from prolonged retention of granules by the melanocyte. This accumulation is not attributable to any visible property of granule structure or formation. The distribution of unmelanized granules within the melanocyte suggests that both Golgi vesicles and free ribosomes are involved in granule formation. The largest melanized granules in all four genotypes are larger than the largest unmelanized granules, but it is not clear whether growth occurs only by increase in thickness of the melanin deposit or whether there is an addition of new framework. I am especially indebted to Dr. Morris Foster for his unflagging contribution of advice, criticism, and mice throughout the course of this study. I am grateful also to Dr. Norman Kemp for advice, assistance, and the use of laboratory facilities, and to Drs. Tahir Rizki, Erich Steiner, and Alfred Stockard for discussion and criticism. I should like to thank Dr. W. C. Bigelow, Mr. John Alley, Mr. Robert Weymouth, and Nancy Smith Istock for advice and assistance in the techniques of electron microscopy, and to thank the photographic department of the Chester Beatty Research Institute for preparation of prints. REFERENCES BIRBECK, M. S. C. (1963). Electron microscopy of melanocytes: the fine structure of hair-bulb premelanosomes. Ann. N.Y. Acad. Sci. 100, 540-547. BIRBECK, M. S. C., and BARNICOT, N. A. ( 1959). Electron microscope studies on pigment formation in human hair follicles. In “Pigment Cell Biology” (M. Gordon, ed. ), pp. 549-561. Academic Press, New York. DOWLING, J. E., and GIBBONS, I. R. (1962). The fine structure of the pigment epithehum in the albino rat. J. Cell Biol. 14, 450474. DROCHMANS, P. (1960). Electron microscope studies of epidermal melanocytes, and the fine structure of melanin granules. J. Biophys. Biochem. CytoZ. 8, 165180. and DROCHMANS, P. ( 1963). Melanin granules: their fine structure, formation degradation in normal and pathological tissues. Intern. Rev. Exptl. Pathol. 2,

357422. FREEMAN, J. A., and SPURLOCK, B. 0. ( 1962). A new epoxy embedment fur electron microscopy. J. Cell BioZ. 13, 437443. Lunr, J. ( 1961). Improvements in epoxy resin embedding methods. 1. Bbphys. Biochem. Cytol. 9, 409-414. MARKERT, C. L., and SILVERS, W. K. ( 1956). The effects of genotype and cell

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environment on melanoblast differentiation in the house mouse. Genetics 41, 429-450. MOYER, F. H. (1963). Genetic effects of melanosome fine structure and ontogeny in normal and malignant cells. Ann. N.Y. Acad. Sci. 100, 584-606. MOYER, F. H. ( 1966). Genetic variations in the fine structure and ontogeny of mouse melanin granules. Am. Zoologist 6, 43-66. RAPPAPORT, H., NAKAI, T., and SWIFT, H. (1963). The fine structure of normal and neoplastic melanocytes in the Syrian hamster, with particular reference to carcinogen-induced melanotic tumors. .I. Cell Biol. 16, 171-186. RITTENHOUSE, E. (1968). Genetic effects on fine structure and development of pigment granules in mouse hair bulb melanocytes. II. The c and p loci, d tlclpp interaction. Deuelop. Biol. 17, 366-381. RUSSELL, E. S. (1949). A quantitative histological study of the pigment found in coat color mutants of the house mouse. IV. The nature of the effects of genie suhstitution in five major allelic series. Genetics 34, 146-166. SEIJI, hl., KIMAO, K., BIRHECK, M. S. C., and FITZPATRICK, T. B. (1963). Subcellular localization of melanin biosynthesiss. Ann. W.Y. Acrid. Sci. 100. 497-5:33. SIXAILE, W. E. (1964). A study of the hair follicle and its mclanocytes. Develop. Biol. 10, 45-70. WELLINGS, S. R., and SIEGEL, B. V. (1959). Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma. J. Ultrastrr~ct. Rea. 3, 147-154. WELLI~\‘GS, S. R., and SIEGEL, B. V. (1963). Electron microscopic studies on the subcellular origin and ultrastructure of melanin. Ann. .N.Y. Acad. Sci. 100,

548-568.