Development 114, 675-680 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

675

Effects of the developmental colour mutations silver and recessive spotting

on proliferation of diploid and immortal mouse melanocytes in culture

EMMANUEL SPANAKIS*, PAMELA LAMINAt and DOROTHY C. BENNETT*

Department of Anatomy, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK

*Present address: Institut d'Oncologie Cellulaire et Moleculaire Humaine, F-93000 Bobigny, FrancetPresent address: Division of Gene Structure and Expression, National Institute for Medical Research, Mill Hill, London NW7, UKtAuthor for correspondence

Summary

The developmental mouse coat-colour mutations silver(si, chromosome 10) and recessive spotting (rs, chromo-some 5, mapping very close to the dominant whitespotting or W/c-kit locus,) appear to reduce the numbersof functional melanocytes in the skin. They were studiedat the cellular level by melanocyte culture. Cellularmorphology, differentiation and survival appeared nor-mal. However, both mutations were found to reduce themelanocyte proliferation rate in primary cultures, asmeasured by [3H]thymidine labelling indices. Two

immortal si/si melanocyte lines (designated melan-siland melan-si2) and one rs/rs line (melan-rs) wereestablished. Melan-sil and melan-rs were cloned. Allthree immortal lines at low passage levels had doublingtimes significantly greater than those of our othermelanocyte lines melan-a, melan-b and melan-c. Thusthey retained the phenotype of slow proliferation.

Key words: coat colour, genetics, silver, recessive spotting,mouse, melanocyte culture, growth.

Introduction

Melanocyte (pigment cell) lines are useful tools formolecular studies of germline coat-colour mutations.After the isolation of normal black mouse melanocytelines by three groups (Sato et al., 1985; Bennett et al.,1987; Tamura et al., 1987) other lines carrying germlinemutations at the b (brown) and c (albino) loci werereported (Abe et al., 1986; Halaban et al., 1988;Bennett et al., 1989). These lines were used to identifythe products of the c and b loci: tyrosinase (an enzymeof melanin pigment synthesis) and a tyrosinase-relatedprotein (Yamamoto et al., 1989; Bennett et al., 1990),respectively. They were also used to characterizemutations at these loci with respect to gene sequence(Jackson and Bennett, 1990), mRNA expression (Jack-son et al., 1990) and protein properties (Halaban et al.,1988; Halaban and Moellmann, 1990).

Coat-colour in mice is affected by mutations at nearly60 loci, more than 20 of which have no effect on eyepigmentation and patchy effects on the coat (Silvers,1979), suggesting that they act on the development ofmelanocytes in the skin rather than on the biosynthesisof pigment. We are characterizing certain of thesemutations, in order to improve our understanding ofthe genetic control of development and perhapsmalignancy. Two such mutations are silver (si) andrecessive spotting (rs).

Silver, although known earlier by mouse fanciers,was first described biologically in 1930, as a mutationthat reduced the number of pigment granules (melano-somes) of the hairs so that some hairs had few andothers none, although some hairs seemed normal(Dunn and Thigpen, 1930). It showed a curiousinteraction with the brown mutation such that the effectof si was greatest on a B/b background. In other words,when homozygous for si, B/b mice were lighter thaneither B/B or b/b mice instead of resembling B/Banimals as usual (ibid.) Our observations confirm this.Later radiological evidence suggested that si/si skin hadreduced numbers of melanocytes rather than a de-ficiency in the synthesis of melanin (Chase and Rauch,1950). Quevedo et al. (1981) reported that si/si hairfollicles had variable numbers of melanocytes whichdied prematurely in the hair cycle. Recently a clonedDNA sequence, pmel 17-1, isolated from a humanmelanocyte cDNA library (Kwon et al., 1987), wasfound to map at or near the murine si locus (Kwon etal., 1991). Expression of the corresponding RNA wasstimulated in melanocytes and melanoma cells byagents that stimulated differentiation (Kwon et al.,1987). However the function of pmel 17-1 is not yetknown.

Recessive spotting was first reported by Dickie (1966)as producing head blazes, large belly spots and diluted

676 E. Spanakis, P. Lamina and D. C. Bennett

bellies when homozygous. There has been no publishedhistological description, but one would expect areduction of melanocyte numbers as observed in otherspotting mutants (Silvers, 1979). These effects re-sembled and interacted with those of heterozygous steel(SI) and dominant white spotting (W allele), (ibid.),and rs was mapped close to W with no recombinationevents so far observed (Southard and Green, 1971;Geissler et al., 1988). It is now known that the W locusencodes the c-kit proto-oncogene, a tyrosine kinasereceptor for a novel growth factor encoded at the SIlocus and given several names including steel factor(SLF), stem cell factor (SCF) and mast cell growthfactor (MGF) (reviewed by Witte, 1990). It has beensuggested that the tight cluster of interacting coat-colour loci on mouse chromosome 5, comprising W, rs,Ph and Rw (Silvers, 1979), may have evolved by tandemreduplication" and divergence. This is supported by thefinding that Ph is a deletion that contains the platelet-derived growth factor receptor A (Pdgfra) gene, whichhas some homology to Kit (Stephenson et al., 1991);moreover another related tyrosine kinase receptorgene, Flk-1, is closely linked to Kit and Pdgfra(Matthews et al., 1991). A syntenic region is found onhuman chromosome 4 (Stenman et al., 1989). Thus thers locus too may encode a tyrosine kinase receptor,whether or not rs is a recessive allele of W or Ph.

Here we describe direct effects of both si and rs onmelanocytes in primary culture and on immortal linesafter establishment and cloning. Both mutations reducethe cellular proliferation rate.

Materials and methods

MaterialsTissue culture media and plastics (Nunc) were obtained fromGibco Europe (Uxbridge, UK) and foetal calf serum (FCS)from Tissue Culture Services (Slough, UK) or Flow Labora-tories (Irvine, Scotland). Cholera toxin, soybean trypsininhibitor and 12-O-tetradecanoyl phorbol-13-acetate (TPA)were from Sigma Chemical Co (Poole, UK). TPA and choleratoxin were dissolved and stored as described previously(Bennett et al., 1985), except that cholera toxin stock solutionwas dialysed to remove azide before storage. The mousekeratinocyte line XB2 (Rheinwald and Green, 1975) wasprovided by J. Rheinwald and adapted by us to grow without3T3 feeder cells as previously reported (Bennett et al., 1989).

MediaA supplemented Eagle's minimal essential medium (SMEM)was used for most purposes. The supplements were penicillin,streptomycin, sodium pyruvate and non-essential amino acids(Kreider et al., 1975) and it was prepared with only 25 mMsodium bicarbonate to give a pH of 6.9 with 10% CO2(Bennett et al., 1987). Unless otherwise indicated, growthmedium was SMEM with FCS at 5% (v/v), and othersupplements as specified.

AnimalsCultures were prepared from embryos or from mice aged lessthan 24 hours. "Wild-type" animals (+/+ at the rs and si loci)were either C57BL/6J inbred mice, Fj hybrids between

C57BL/6J and LAC-MF1 mice, or Fj hybrids betweenC57BL/6J and CBA/Ca mice. The si mutation was studied inanimals heterozygous for b (see Introduction), obtained bycrossing si/si B/B and si/si b/b mice, si/si B/b and rs/rs mice,both of a C57BL/6J background, were obtained from theJackson Laboratories, Bar Harbor, Maine. All breeds weremaintained at St George's Hospital Medical School.

Primary cultures of melanoblastsSome cultures were prepared as described previously (Mayerand Oddis, 1977; Bennett et al., 1989). More recently thetechnique was modified to increase the yield of melanocytesand distribute them more homogeneously in culture, asfollows. Neonatal mice were killed, washed in 70% ethanolfor approximately 10 seconds and then in Dulbecco'sphosphate-buffered saline lacking calcium and magnesiumchlorides (PBSA). Trunk skins were incubated with 5mg/mltrypsin in PBSA at 37°C for 1 hour, then transferred to aculture dish containing PBSA. Epidermal sheets wereremoved, pooled in 100 /il of PBSA with trypsin (250 /ig/ml)and EDTA (200 j/g/ml) on another plate, and minced finelywith a scalpel. The tissue was homogenised in 5 ml SMEMcontaining 200 ng soybean trypsin inhibitor, by vigorouspipetting with a 2.5 ml Combitip (Eppendorf, Hamburg) anddiluted into growth medium (about 10 ml/donor mouse). Thesuspension was supplemented with cholera toxin (1 nM) andtransferred to plates containing mitomycin-treated XB2keratinocytes as feeder cells. These were plated 1-3 daysearlier as described previously (Bennett et al., 1989). TPA(200 nM) was added at the first medium change (1-3 days).Further procedures were as before (ibid.).

Subculture and cloningUnless otherwise stated, non-immortal melanocytes weregrown in SMEM with 5% (v/v) FCS, TPA (200 nM) andcholera toxin (1 nM), while immortal rs/rs and si/si melano-cytes were grown in the same medium except with 10% FCSand no cholera toxin. For subculture, cell suspensions wereprepared from growing cultures with trypsin (250 /ig/ml) andEDTA (200 /igrnil) as described (Bennett et al., 1987), andwere replated at 3-5 x 104 cells/ml. Early subcultures weremade on to fresh XB2 feeder cells. If cultures became verysparse, Ham's F10 medium with 18 mM bicarbonate wassubstituted for SMEM. For cloning, manually selectedindividual pigmented cells were transferred to separate wellsof a 96-microwell plate, each containing XB2 feeder cells and100 fA growth medium conditioned by them (Bennett et al.,1989).

^HJthymidine autoradiographyThe culture medium was supplemented with [3H]thymidine (1/iCi/ml) (Amersham) and inosine (25 /iM) (Brooks, 1977) for48 hours. The cells were then fixed with 4% formaldehyde inDulbecco's complete PBS for 10 minutes, extracted with ice-cold 5% (v/v) trichloroacetic acid in water for 1 minute,dehydrated by 3 washes with absolute ethanol and air-dried.Plates were coated with chrome alum-gelatin solution, air-dried and coated with Ilford K5 nuclear emulsion (50% v/v inhot water), exposed and desiccated at room temperature for 3days, and developed.

The plates (usually 3 per treatment) were coded. Labelledand unlabelled nuclei in at least 3 samples of at least 100pigmented cells were counted per plate, from randompositions. The percentage of labelled nuclei was calculated for

Mouse mutations and melanocyte growth 611

each plate, and arithmetic means and standard errors for eachtreatment were determined after decoding.

Doubling timesCrude population doubling times were obtained duringroutine passage of cell lines, which were not allowed tobecome confluent. Cells were plated at a known density anddoubling times were calculated from the number of days'growth and the mean cell density attained, as measured by atleast 3 independent haemocytometer counts of at least 150cells each. Melan-a, melan-b and melan-c melanocytes (black,brown and albino) were grown as previously described(Bennett et al., 1987, 1989).

Fig. 1. Appearance of mutant melanocyte cultures. Toillustrate melanocytes alone, immortal clones rather thanprimary cultures are shown. Cultures were fixed withformalin, stored dry and water was put in the dishes forphotography. Phase contrast optics. (A) rsjrs (melan-rs)cells. Several giant cells are visible. (B) si/si (melan-sil)cells, grown with XB2 keratinocyte feeder cells until theprevious passage. At least one residual XB2 cell is visible(double arrowhead). Small, highly-refractile cells are thosein mitosis. (C) Wild-type melanocytes (melan-a), growingwith only 5 nM TPA to illustrate slowly-growing cells. Asin A and B, melanocytes are flattened and somewhatheterogeneous; the morphology is intermediate betweenthe spindly cells seen with 200 nM TPA and the polygonalquiescent cells without TPA (see Bennett et al., 1987).(Note that the darkness of pigmentation cannot becompared directly with A and B, as C was photographedon a different occasion and the optics may have differed.)Bar, 300 fan.

Results

General phenotypes of si/si and rs/rs melanocytes incultureOnly unpigmented melanoblasts are initially obtainedfrom neonatal or embryonic mouse epidermis, togetherwith keratinocytes, but melanoblasts become pig-mented within about 2 weeks under the given con-ditions, while keratinocytes are gradually lost. Visualinspection of the resulting cultures by microscopyrevealed no obvious differences between wild-type andmutant melanocytes of either type, in their shape, size,migration or differentiation, the last being assessedfrom the time of appearance of pigment. The appear-ance of melanocytes of both mutant genotypes is shownin Fig. 1. Their resemblance to other melanocytes wasgreatest where the latter were growing slowly (Fig. 1C),whereupon the cells became more flattened andheterogeneous, with occasional giant cells even inimmortal cultures (Fig. 1A; not shown in Fig. 1C).Giant cells are also common in senescent diploidmelanocyte cultures of any genotype.

On further culture of the diploid cells, it becameapparent that si/si and rs/rs melanocytes were notreaching confluency as soon as wild-type cultures,suggesting slower growth and/or a higher death rate.Melanocyte proliferation in primary cultures wasquantitated by [3H]thymidine autoradiography of pig-mented cells. This method was chosen because total cellcounts and other measures could be affected by celldeath rates or by contaminating non-pigmented cellpopulations.

In Table 1, melanocyte proliferation in cultures fromanimals of the three genotypes is compared, in terms ofthe percentages of melanocytes labelled in 48 hours.Results from cultures of various ages between 3 and 5weeks (after plating) were pooled from 13 experiments.Cells of all genotypes showed a decline in DNAsynthesis with the age of the culture. This decline was atleast partially associated with increased cell density;larger colonies showed reduced labelling, especially in

678 E. Spanakis, P. Lamina and D. C. Bennett

Table 1. Proliferation rates of diploid melanocytes of 3 genotypes in primary culture

Day of culture

Genotype 17 19 20 21 22 23 24 27 28 33

/si/sirs/rs

50±8 41±250±4

57±436±429±5

5139±340±2

56±8

36±3

50±537±3 39±5 30±3

20±218±1

3640

Values show the thymidine labelling index of pigmented melanocytes, mean % ±s.e.m. Primary cultures were prepared from embryonicor neonatal epidermis and processed for [3H]thymidine autoradiography as described (Materials and methods). The labelling period was 48hours, ending on the specified day. Data are pooled from 13 independent experiments. Cultures of both mutant types showed highlysignificant differences from +/+ cultures by analysis of variance: /3=0.0011 for rs/rs, P<0.00005 for si/si.

their centres where crowding was highest (not shown).However, si/si and rs/rs diploid melanocytes showedthis decline at earlier times (and thus lower populationdensities), as can be seen from Table 1. Overall, thedifference in labelling index of melanocytes of bothgenotypes compared with wild-type cells was highlysignificant by analysis of variance (Table 1).

Establishment and growth rates of immortal linesTwo melanocyte lines were isolated at different timesfrom independent si/si B/b cultures and one from anrs/rs culture; these were designated melan-sil, melan-si2 and melan-rs respectively. The methods used weresimilar to those described previously (Bennett et al.,1987,1989), though the time taken for the emergence ofreliably growing, established cultures was long (8months for melan-rs, 6 months for melan-sil, notrecorded for melan-si2). As before, all three linesretained a requirement for TPA in the mediumdescribed, although cholera toxin was omitted oncecontaminating cell populations were eliminated.Melan-sil and melan-rs were cloned with high ef-ficiency. The slow proliferation from both genotypescontinued throughout establishment and at least inearly passages subsequently, although growth acceler-ated at high passage levels as seen with all ourmelanocyte lines (e.g. Bennett et al., 1987). Doublingtimes of the lines melan-sil and melan-rs at low passagelevels are compared with those of other establishedmelanocyte lines in Fig. 2. Both showed slowerpopulation growth than any previous line, with doub-ling times 3-4 times greater in medium with 10% FCSthan melan-a black melanocytes which have the samegenetic background.

There may have been a contribution of increaseddeath rate to the increased doubling times, but thiscannot have been major, as dead melanocytes can bedetected either as floating black cells or by the releaseof black melanosomes into the medium on cell lysis; andvery little death was observed in these lines under thestandard conditions. Thus reduced proliferation musthave accounted for most of the reduced populationgrowth.

Other propertiesBoth melan-sil and melan-si2 cells, when newlyestablished, showed an unusually marked dependenceon feeder cells for reattachment and survival after

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Fig. 2. Crude doubling times of immortal melanocytes of 5genotypes, obtained as described (Materials and methods).Each point indicates one measurement. (O) in 10% FCS;( • ) in 5% FCS; other supplements as usual. 2-mercaptoethanol (100 /iM) was present in cultures whosegrowth was improved by it, namely lines melan-b andmelan-c (see Bennett et al., 1989). a, melan-a; b, melan-b;c, melan-c; rs, melan-rs; si 1, melan-sil.

subculture, particularly after frozen storage and thaw-ing, or following more than one subculture withoutfeeder cells (Fig. 3). One day after the illustratedcultures were plated, most of the melanocytes appearedto be unattached and dead in the plates without feedercells, whereas very few floating melanocytes were seenin those with XB2 cells. This suggested the possibility ofalteration in an extracellular attachment (matrix)factor, or a receptor for one. However the effect waslost at later passages, presumably by selection of cellscapable of better attachment, and thus could not becharacterized further. It may have been a quantitativerather than qualitative difference from other genotypes,perhaps reflecting the general level of health of thecells, as melanocytes of all genotypes grew considerablybetter in the presence of XB2 cells before immortaliza-tion.

Mouse mutations and metanocyte growth 679

Fig. 3. Effect of feeder keratinocytes on melan-sil cells.After 1 passage in the absence of feeder cells, cultureswere trypsinized again and plated without (A) or with (B)feeder cells. Plates were fixed for photography after 11days' culture. Magnification and optics as in Fig. 1.Keratinocytes (arrowheads) can be identified in B by theirlarge, pale nuclei surrounded by a ring of pigment (derivedfrom the melanocytes).

Discussion

Both si/si and rs/rs melanocytes grew more slowly thanthose of other genotypes, both in primary culture andafter immortalization. Both mutations were on thegenetic background of C57BL/6J, but the possibilitythat the slow growth was a result of this background canbe ruled out by previous observations that melanocytesfrom this strain of mice grow well (Sato et al., 1985;Bennett et al., 1987): as well as or better than thosefrom other strains (Tamura et al., 1987). The si/si andrs/rs melanocytes otherwise appeared generally normal,except for a possible deficiency of attachment in si/simelanocytes. These findings are consistent with thepostulation that the rs mutation affects a growth-factorreceptor, while not proving this. If rs is indeed allelicwith W/Kit, it may affect a part of the SCF receptorgene other than those altered by the dominant Wmutations (Nocka et al., 1990), such as that encodingthe extracellular domain, or a control sequence regulat-ing expression. If the rs locus encodes a differentreceptor, two candidates are the PDGF receptor A and

Flk-1 (Stephenson et al., 1991; Matthews et al., 1991;see Introduction). We do not know of evidenceseparating the rs and Ph loci, although rs is distinct fromRw because rs maps inside the W deletion and Rwoutside it (Geissler et al., 1988).

Reduced proliferation is also consistent with theobserved phenotype of reduced numbers of hair-follicular melanocytes in si/si skin. No major increase indeath rate was observed in culture, but this is notincompatible with the report of premature death ofsilver melanocytes in the hair cycle in vivo (Quevedo etal., 1981). This death may depend on periodic eventsrelated to the hair cycle, such as falling levels of growth-or survival-factors, or increasing melanocyte differen-tiation, which may not occur in culture. Mintz (1971)suggested - from the patterns of pigmentary mosaicismin chimaeric mice containing both wild-type and si/sitissue - that the silver mutation acted primarily on thefollicles rather than the melanocytes. We however haveobserved a direct effect on cloned melanocytes. It ispossible that the si gene product is a growth, survival orattachment factor synthesized both by melanocytes andby other skin cells, so that si melanocytes can functionnormally in wild-type skin (as implied by Mintz'sreport), and wild-type melanocytes in mass culture canalso sufficiently provide the factor, but isolated simelanocytes cannot. This would not however explainthe interaction of the b mutation with silver. Theproduct of the b locus is a presumptive melanosomalenzyme, tyrosinase-related protein 1 (TRP-1) (Jacksonet al., 1990; Bennett et al., 1990), which alternativelysuggests a melanosomal location for the si geneproduct. Moreover the product of pmel 17-1, themelanocyte protein that is a candidate for the si locusproduct, shows areas of sequence homology with TRP-1, the b locus product, and with tyrosinase (Kwon et al.,1991). This suggests that it may be another melanoso-mal enzyme, which could easily interact with TRP-1 in acompetitive manner. It may seem surprising that amutation in a pigmentary enzyme could inhibit cellgrowth, but for comparison there are two known b-locus mutations that actually kill melanocytes as theydifferentiate in each hair cycle (Jackson et al., 1990).

We and others are now investigating the moleculardefects present in rs/rs and si/si melanocytes, by variousmeans including analyses of the integrity of parts ofgrowth-signalling pathways that could be cell-typespecific, and of the state of c-kit in rs/rs melanocytes andpmel 17-1 in si/si cells. The cell lines reported hereshould greatly assist the characterization of these twogenes required for the development of a full comp-lement of melanocytes in the skin.

This research was supported by the Wellcome Trust and theCancer Research Campaign. We thank numerous colleaguesfor stimulating discussions and Byoung Kwon for communi-cation of unpublished work.

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(Accepted 28th November 1991)