TIG - - December 1985

2 Degnan, S. T. and Newton, A. (1972) Chromosome repli- cation during development in Caulobacter ~ t u s . ]. Mot Blot 64, 671-80 3 Shaw, P., Comes, S. L., Sweeney, K., Ely, B. and Shapiro, L.

(1983) Methylation involved in chemotaxis is regulated during Caulobacter differentiation. Pn¢_. Natl Acad Sd. USA 80, 5261 4 Comes, S. L. and Shapiro, L. (1984) Differential expression

and ix~sitiorting of chenmtaxis methylation proteins in ~ b a c t e r . J. Mot Biol 178, 551-568

5 Johnson, R. C., Walsh, J. P., Ely, B. and Shapiro, L. (1979) Flagellar hook and basal complex of Caulobacter crescgntus, f Bac- ter/ol. 138, 984 6 Stallmeyer, M. J. B., De Rosier, D. J., Aizawa, S.-I., Macnab,

R. M., Hahnenberger, K. and Shapiro, L. (1985) Structural Studies of the basal body of bacterial flagella. Biophys. f , 47-48a

7 Wagenknecht, T., De Rosier, D., Shapiro, L. and Weissborn, A. (1981) Three-dimensional reconstruction of the flagellar hook from Caulobacter crescentus. ]. Mot Biol. 151,439 8 Koyasu, S. and Shirakihara, Y. (1984) Caulobactercrescentus

llagellar filament has a right-handed helical form.]. Mot BioL 173, 125 9 Gil, P. R. and Agabian, N. (1982) A comparative structural

analysis of the flagellin monomers of Caulobacter crescen/us indicates that these proteins are encoded by two genes. J. Bacte'n;o/. 150, 925 I0 Weissborn, A., Steinman, H. M. and Shapiro, L. (1982) Characterization of the proteins of the Cgulobacter crescentus flagellar filament: peptide analysis and filament organization. ]. Blot Chem. 257, 2066 11 Fukuda, A., Asada, M., Koyasu, S., Yoshida, H., Yginuma, K. and Okada, Y. (1981) Regulation of polar morphogenesis in Caulobaaer ~ t u s . J. Bacterlo/. 145, 559 12 Johnson, R. C., Ferber, D. M. and Ely, B. (1983) Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants. J. Bacter',bl. 154, 1137 13 Ely, B., Croft, R. H. and Gerardot, C. J. (1984) Genetic map- ping of genes required for motility in Caulobacter crescentus. Gene- t ~ 108, 523-532 14 Osley, M. A., Sheffrey, M. and Newton, A. (1977) Regulation of flagallin synthesis in the cell cycle of Caulo&u'~. dependence on DNA replication. Cell 12, 393-400 15 Lagenaur, C. and Agabian, N. (1978) Caulobacter flagellar organeUe: synthesis, compartmentation and assembly.]. Bactenbl. 135, 1062 16 Ohta, N., Chen, L.-S. and Newton, A. (1982) Isolation and expression of cloned hook protein gene from Caulobacter cres- centus, t~,oc. Natl Acad. Sci. USA 79, 4863-4867

review 17 Purucker, M., Bryan, R., Amemiya, K., Ely, B. and Shapiro, L. (1982) Isolation of a Caulobacter gene cluster specitying flagel- lum production by using non-motile Tn5 insertion mutants. Pr~. Natl Aaut. Sci USA 79, 6797-6801 18 Mil_hausen, H., Gil, P. R., Parker, G. and Agabian, N. (1982) Cloning of developmentally-regulated llagellin genes from Caulo- batter crescem~ v/a immunoprecipitation of polyribonomes. ]Woc. Natl Acad, Sci. USA 79, 6847-6851 19 Milhausen, M. and Agabian, N. (1983) Cmdobacter flagellin mRNA segregates asymmetrically at cell division. Nature 302, 630qi32 20 Champer, R., Bryan, R., Comes, S. L., Purucker, M. and Sbaprio, L. Temporal and spacial control of flagellar and chemo- taxis gene expression during Caulobacter cell differentiation. Co/d Slm'ng Harbor Syrup. Quant BioL (in press) 21 Newton, A., Ohta, N., Huguenel, E., Chen. L.-S. (1985} in Spores IX (Setlow, P. and Hoch, J., eds.) Am. Soc. Microbiol. (in press) 22 Bellofatto, V., Shapiro, L. and Hodgaon, D. (1984) Genera- fion of a Tn5 promoter probe and its use in the study of gene expres- sion in Caulobaaer crescentus. Proc. NatlAcad Sci. USA 81, 1035 23 Bryan, R., Purucker, M., Comes, S. L. Alexander, W. and Shapiro, L. (1984) Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in ~ crescent~ using plasmid complementation. Proc. Natl Acad. Sci USA 81, 1341 24 Ohta, N., Swanson, E., Ely, B. and Newton, A. (1984) Phy- sical mapping and complementation analysis of Tn5 mutations in Caulobacter crescentus: organization of transcriptional units in the hook cluster, f Bacter/ot 158, 897 25 Komeda, Y. (1982) Fusions of flagellar operons to lactose genes on a Mu loc bacteriophage. J. Bacter/ot 150, 16 26 Agabian, N., Evinger, M. and Parker, E. (1979) Generation of asymmetry during development. ]. Cell Blot 81, 123-36 27 Lagenaur, C., Farmer, S. and Agabian, N. 0974) Absorption properties of stage-specific Caulobacter OCbk. Virology 77, 401-407 28 Huguenel, E. D. and Newton, A. (1982) Localization of sur- face structures during prokaryotic differentiation: role of cell divi- sion in Caulobacter ~ t u s differentiation. Lhfferentiation 21, 71-78

L Shapiro is at the Department of Molecular Biology, Division of Biological Sdences, Albert Eins~'n College of Med~'ne, Bronx, N Y 10461, USA.

In the mouse , me lanocy tes (the p igment -p roduc ing cells) have an in teres t ing embryological history, or iginat ing in the neural c res t of the embryo and subsequen t ly migra t ing to colonize the skin. W h e n in the hair follicle, me lanocy tes can p roduce both yel low phaeo- melanin and black eumelanin, the p igmen t s which colour mouse hair. T h e de rmal cells of the hair follicle regula te syn- thes is and deposi t ion of the pig- m e n t s by the melanocytes , swi tching syn thes i s b e t w e e n eumelanin and phaeomelan in so that in the wild-type mouse the dorsal hairs have a black tip and black base sepa ra ted by a yellow band. T h e ventra l coat of the wild-type mouse appears l ighter because s o m e hairs lack the banded pat tern , ins tead hav ing simply a yel low tip and black base. Owing to the i r rich genet ic diversi ty, the coat colour g e n e s have rece ived much at tent ion, and I have not a t t e m p t e d a comprehens ive coverage of the field (for this see Silvers, Ref. 1). Ins tead, I highl ight some of

Genetics and biology of mouse melanocytes: mutation,

migration and interaction lan J. Jackson

Coat coloration in the mouse provides a convenient assay for new mutations and a model for complex g e m interactions. The interesting embryological behaviour o f melanocytes (pigment-producing cells) serves as a good model for cell migration during development and the action o f genes on this migration. Genes closely linked to coat colour genes have been found to have speafic effects

in development.

the more in teres t ing aspects , part icularly w h e r e recen t p rogress has been made .

I focus on th ree well s tudied loci. T h e albino (C) locus on c h r o m o s o m e 7 has seven well charac ter ized alleles and m a n y radiat ion- induced mutat ions , mos t of which show pleiotropic ef fec ts on embryonic develop- m e n t as a result of delet ions of ne ighbour ing genes . Muta t ions at C reduce amounts of both p igment types. T h e agouti (A) locus on c h r o m o s o m e 2 has over 15

© 1965. Elnevier S, cien¢~ ~ B.V.. Anu~rdam 0168 - ~ . 0 0

r views ( r ~

( b )

Fig. 1. (a) Chimaeric mice made by mmN'ning embryonic cells of a pigmented strain with an unpigmented strairt Note discrete patches of colour, and bilateral asymmetry of dism'bution. Chimaeras produce offspn'ng of both parental strain genotylPes. (b) A p~/ptm male, showingextensive mo.~icism, with two offspring produt~d from a cross to a pip female. One offspring is of the 'low- grade mosa/c' p~/p type, the other is apparently pi +.

15 alleles or pseudoalleles, which regulate the balance between black and yellow pigment deposition in the hairs over different parts of the body. The dilute (D) locus maps to chromosome 9. It has only a few alleles among the coat colour mutations, some of which pro- duce neurological defects in addition to the lightening effect on the pigmentation of the hair. DNA from the D locus has been isolated as recombinant bacterio- phage, which has permitted some correlation between the genetics and the gene structure.

Embryology of coat colouration The embryonic history of the melanocyte prompted

some workers to call them 'honorary neurones' and the cells do indeed have a morphology reminiscent of nerve cells. The melanocytes of the skin originate in the neural crest z. Cells migrating away from the crest differentiate to several cell types. The cells migrating near the surface become melanocytes, whilst other cells, migrating deeper, form parts of the peripheral nervous system and the cells of the adrenal medulla. In the head area, cells of the neural crest also form parts of the craniofacial skeleton. The hair follicle is made up of components from two of the primary germ layers of the embryo, mesoderm (the dermis) and ectoderm (the epidermis, from which the hair forms). Melano- cytes enter the hair follicle as it develops from an

T I G - December 1985

invagination of the epidermis into the dermal tissue beneath and they secrete melanin granules into the hair as it grows. After growth, the follicle enters a quiescent phase and melanocytes are no longer visible. Plucking a hair stimulates the reappearance of melanocytes, which either migrate in from nearby skin or divide and differentiate from resident stem cells.

The pattern of pigmentation in chimaeric mice produced by combining embryos of a coloured strain with an albino strain provides a striking illustration of melanoblast migration. Pigmentation occurs in patches, usually stripes formed asymmetrically about the dorsal midline (Fig. la). This shows that the pig- ment in a particular patch arises from one or a few melanoblasts which have only limited migration once established; clonal descendants migrate to colonize one area, each clone being restricted to one side of the midline. Mintz suggested 3 from an examination of chimaeras that there are 34 clones along the length of the mouse, 17 on each side. Although there is not general agreement about this figure, the number of stem cells giving rise to melanocytes must be small to produce a patchy rather than a uniformly brindled coat'. Each hair follicle may contain up to 20 melanocytes, not necessarily derived from the same precursor melanoblast. Between pigmented and unpigmented patches in a chimaeric or mosaic mouse, where hairs of both pigment types mingle, some hairs have non-uniform pigmentation. This is because one follicle is populated by both parental types of melanocyte 5.

Mutations at several loci affect the migratory ability of the melanoblasts. Some (notably dominant white spotting) also affect the migration of other cell types, in particular germ cells and haematopoietic cells.

S i te of gene action The migratory properties of melanocytes have been

used to localize the cellular site of coat colour gene action. A fragment of skin from a mouse with melano- cytes producing no or very little pigment (albino, c/c, or extreme dilution, c'/c ') when grafted to a wild-type newborn mouse, is invaded by wild-type melanocytes from the surrounding host tissue to produce coloured hairs. The phenotype of these hairs is determined by the genotypes of both the host melanocytes and the graft hair follicle. Whilst most mutations only affect phenotype through the melanocyte, mutations at the agouti locus are expressed in the hair follicle.

Two very different phenotypes produced by mutation at the agouti locus are yellow (the dominant mutation A r) and non-agouti, an almost completely black phenotype when melanocytes are wild-type (the recessive mutation a). Skin from a mouse of genotype a/a c'/c ~ normally grows pale brown hairs. However, when grafted onto an AY/a C/c" host, which has yellow hairs, the graft grows a few black hairs. This is because melanocytes from the host migrate into the graft and produce black pigment in response to the non-agouti skin s (Fig. 2a). Conversely when the very pale hairs and skin of an AY/a c'/c" mouse are grafted onto a black host of genotype a/a C/c' the host melanocytes which migrate into the graft, although they produce black pigment in the host, cause yellow hairs to be produced in the graft 6 (Fig. 2b). Thus the

TIG - - December 1985

albino product is expressed in melanocytes, whilst agouti expression is in the hair follicles in the skin. By separating the mesodermal from the ectodermal com- ponents of skin from embryonic mice and recombin- hag them with components of genotypes differing at the agouti locus it can be seen, after culture in a suit- able host, that the hair colour which develops is largely determined by the agouti alleles of the dermis (meso- derm) s-s.

Bowman and McLaren s used chimaeric animals to study this interaction between melanocyte and fol- licle. They showed that mutations at the four loci: chinchilla (an allele of albino, c'~), brown (b), dilute (d) and pink-eyed dilution (p), are expressed in the melanocyte whilst the non-agouti (a) mutation determines phenotype via the hair follicle. Thus, chimaeras between embryos homozygous for these five recessive mutations and embryos wild-type at those loci give rise to mice with patches of hair of three different colours. Where 'recessive' melanocytes occupied recessive or wild-type skin the hair was cream, the colour of the mutant parental type. Wild- type melanocytes in wild-type skin produced wild- type hairs. The third type of patch was black, where wild-type melanocytes interacted with hair follicles of the mutant type. This black pigmentation results from the expression of the non-agouti mutation in the follicle.

Agouti locus The agouti locus is the most genetically complex of

the coat colour loci. It acts in the hair follicle to switch pigment synthesis in the melanocyte between the black and yellow pigment types. That individual melanocytes can switch is indicated by the identifica- tion of a few melanocytes containing both pigment types, caught in the act of switching s. The agouti product is presumed to be some type of signal to the melanocytes about which pigment (yellow or bladrJ.to make, so that during hair growth the signal acts to produce a yellow strip between the black tip and base of the hair shaft. The signal can be mimicked, at least in part, by melanocyte-stimulating hormone (MSH). Yellow (AY/-; C/C) mice, which normally produce no black pigment will do so locally in response to an injec- tion of MSH ~°. Skin explants in culture respond to MSH in the same way n. The mutation recessive yellow (e) is expressed in the melanocyte and seems to identify the receptor of the signal on this cell. Melanocytes from mice which are e/e produce only yellow pigment as do melanocytes in AY/- skin but unlike those from AY/- animals theywill not respond to MSH. However, the e/e melanocytes do respond by producing black eumelanin after treatment with dibutyl cAMP, which is possibly bypassing the receptor and acting intracellularly to activate eumelanin synthesis z2.

The 17 alleles at the agouti locus can be arranged in a dominance series. The 'top' dominant, lethal yellow (.40 produces a yellow coat in combination with any other allele, whilst at the other end of the series, extreme non-agouti (a'), when homozygous, produces a completely black coat. These represent the extremes of possibilities for the melanocyte. The other alleles in the series, moving down from AY,

review produce mcreasi~ly darker dorsal hairs, wl~lst the bdly hairs remain light. Some alleles recessive to wild- type, when made homozygous, have completely black hairs on the back (with no yellow bands) but have Light belly hairs. This differential behaviour of hairs on different parts of the body has led some workers to suggest that the genes at agouti form a complex of several 'mini-loci' exhibiting pseudoalleLism, in which different genes regulate colouration of the back, belly a n d s o o n 13.

Although there is no direct evidence for mini-loci of this type, pseudoallelism is clear at the locus. Two mutations, yellow (.4:') and lethal non-agouti (a0 map to the agouti complex and affect its expression. Both are recessive lethal genes, but affect different developmental functions when homozygous. A' /A y embryos are abnormal at implantation and die soon afterwards 1~ whilst aVa ~ embryos die earlier than this as. Fuahermore, the mutations complement each other so that A'/a ~ mice are viable. The balanced lethal stock, AY/a x x Aqa" produces phenotypically wild- type offspring at a frequency of about 1/500 (Ref. 16). Flanking markers indicate that these are recom-

~k' ~ " 4 / I ~10,,, 11 (a) black

du~nis

( b ) Graft Holt

nmkmocy~s

<

i dermis o o

Fig. 2 Schematic representation of sections through the skin of grafted nude. Graft/s on the/eft, hast on the ~ighL (a) M/a C/c' grafted m'~ a a/a ce/c e host," ~) a/a C/e e ~'aft~ on~ a AY/a ce/c e host. The C-locus genoO~ of melanocytes and A-locus genotypes of dermis, and colours of hairs seen are shourtt Migration of melanoeytes from host to graft is indicated by the artmos. 3 2 ~

v i e w s E t W.T. 9kb

E E d ' ~ / 18 kb

E d ÷ i 9.3kb Fig. 3. Insertion of MuL V DNA at the dilute locus. Only EcoR1 ~ sites are shown, and the size of the target fragment is on the right. The heavy lines represent the viral long terminal repeats (L TR.q, and viral DNA is enclosed between thent W. T. = wild- O~Oe DNA; d = dilute mutation DNA; d + = DNA from a retertant of dilute to wild type. Data from Ref. 21.

binants and not revertants. Thus there seem to be two closely linked genes (about 0.5 cM apart) which are essential for embryonic development, and have mutations affecting coat coloration via expression of the agouti 'gene'. These may both be genes required for full expression of the agouti phenotype, whose products are also required for development of the embryo. On the other hand, it is possible that the lesions are in genes unrelated to agouti, but the mutations exert an effect in c/s over a distance on the chromosome.

Another mutation of the agouti complex, Agouti suppressor (A~), clearly acts in this way. A ' exhibits a c/s-dominant effect to lighten the phenotype of the linked agouti allele ~7. Recombination between A ~ and the agouti alleles is 0.6%. The nature of the suppres- sion is not known, and is confused by the observation that this X-ray induced mutation is associated with a chromosomal inversion, extending from at or near the agouti locus for 40% of the length of chromosome 2 (Ref. 18). Perhaps chromosome location of the agouti gene affects its expression. Given the recombination between A y and a¢ in addition to the recombination

imptant - ;mr lna ta l C runtod Mod-2 - I "1 -2

Fig. 4. Comp/ementatfi~n groups around the C/ocus, defined by analysis of albino deletions. Rearmbination distances betuzen C Mod-2 and sh-1 are shour~ Most possible combinations of delet/ons, enarmpass/ng C plus add'n/rig groups have teen observed Mod-2-mitochondrial malic enzyme; sh-1 - shaker-l; preimplant - homazyg~,s embryos with this deleted die before implantation; implant-l, imlgant-2 - homazygom embryos with either gro~ deleted die soon after i ~ " ~ - mice homozygous for a deletion of this g~Tup die soon after birtk They show ~rphological ab~,nah'ties of the liter and deficiencies in some liver and serum emymex hinted - mice hom~ygous for a deletion of this group and runts at birth, are sterile and usually die

3 ~ before 4 months of age. (See Ref. 28.)

T I G - December 1985

with A ~, mutations spanning >1 cM can affect agouti expression. This could indicate a physical distance of many hundreds of kilobases, although the correlation between physical distance and recombination fre- quency may break down at such short genetic distances.

Many questions about the physical nature of the agouti locus should soon be answerable. Copeland and Jenkins have observed that the Ay mutation is closely associated with an ecotropic leukaemia provirus 19. Southern blots reveal a 4.7 kb !h,uII fragment of MuLV which cosegregates with A y through many backcrosses and sib-matings. Without revertants it is not possible to prove that the proviral insertion causes the A y mutation; however, the close genetic asso- ciation means that the provirus can be used as a molecular probe to isolate DNA from this region. It should then be possible to resolve some of the complex genetic issues by analysis at the DNA level. In addi- tion, the use of DNA probes will allow identification of embryonal stem cell lines homozygous for the lethal alleles, which will facilitate investigation of the de- velopmental effects of these alleles.

Dilute locus Compared with the A-locus, most other coat colour

loci seem simple. In molecular terms the best under- stood is the dilute locus, as this has been isolated as a cloned DNA fragment. The dilute mutation (d) reduces colouration in the coat by altering the distribution of melanin granules within the melanocyte. Granules in wild-type melanocytes migrate out into the dendrites from where they are deposited into the hair. In dilute melanocytes the granules cluster around the nucleus and are deposited in the hair in clumps, so that although the pigment volume is the same as in wild-type hair the colouration is reduced by the uneven distribution. The product of the wild-type dilute gene is possibly a cytoskeletal element involved in the distribution of the melanosomes.

Jenkins and Copeland showed by Southern blot hybridization that the dilute mutation was associated with a MuLV proviral insertion 2°. Since the d mutation reverts to wild-type at a frequency of 10 -5 to 10 -6, it was possible to examine these revertants and show that reversion is accompanied by loss of proviral DNA 2°. The provirus was used to clone the d DNA, which was in turn used to isolate the wild-type and revertant genes2L Closer examination showed that on reversion the proviral excision left behind one of the long terminal repeats (LTRs) which normally flank the provirus. The reversion presumably occurs by recombination between the terminal repeats, causing a deletion of the enclosed viral sequence from the chromosome (see Fig. 3). The presence of the 9 kb provirus disrupts gene expression and produces the dilute phenotype, whereas the 400 bp LTR in the revertants does not. It is not surprising, then, that sequencing the site of proviral integration reveals no open reading frames 22. It is not known whether the insertion is into an intervening sequence or in flanking DNA, but transcriptional studies, probing RNA from melanoma cells, will answer this.

Radiation-induced deletions of the dilute locus have been made at the Oakridge National Laboratory,

T I G - December 1985

USA. These probably span different amounts of DNA in addition to the dilute locus and delete several closely linked genes essential for growth and development of the embryo. Some encompass the short-ear locus, a mutation about 0.18 cM from dilute, which causes homozygotes to have small ears. It should be possible, using large cloned fragments, in conjunction with the deletions, to walk to, clone and identify the short-ear gene and other linked genes. The d-se complex has been reviewed recently in Trends in Genetics ~.

Several alleles of d produce neurological defects. The most severe in the series, dilute-lethal, in addition to its effect on coat colotwation, causes death at three weeks from myelin degeneration in the central nervous system. Other alleles of d have a less severe neurological defect. It will be interesting to determine whether the neurological defect is due to a pleiotropic effect of the coat colour gene itself, or to action on a closely linked gene.

The only known recessive suppressor gene in the mouse, dilute suppressor (dsu), acts at this locus 24. When dsu (which maps to chromosome 1) is homo- zygous, the lightening effect of the homozygous d mutation on the coat is totally suppressed, although the provirus remains integrated in the chromosome. Moreover, in combination with dilute lethal, dsu sup- presses the coat colouration effect but not the neuro- logical defect. The action of dsu is probably to act on melanocytes independently of d, because it also will suppress the lightening of coat colour due to ashen, a mutation mapping close to, but clearly separate from, dilute.

Albino locus Mice homozygous for the albino mutation (c)

produce no pigment. Other alleles of c have reduced amounts of both pigment types. Biosynthesis of the two pigments begins from tyrosine, which is hydroxy- lated by the enzyme tyrosinase to dihydroxy- phenylalanine (DOPA). Tyrosinase also oxidizes DOPA to dopaquinone, from which either eumelanin is made by conversion to indole forms before polymerization or phaeomelanin is synthesized by addition of dopaquinone to cysteine and poly- merization. The only enzyme used in both pigment pathways is tyrosinase, and this is thought to be encoded at the albino locus.

Measurements of tyrosinase activity in skin slices show that it is almost absent from albino skin, and is correlated with the extent of pigmentation in mice with other alleles 25. Skin from mice with the Himalayan mutation (c*) shows temperature sensi- tivity with respect to tyrosinase zS. This correlates with the phenotype of the mouse, which has a white body but dark ears, nose and tail. However, these experi- ments assayed tyrosinase in intact tissue and may not be measuring the enzyme itself but rather another aspect of melanocyte function which indirectly affects tyrosinase activity.

Antisera against mouse tyrosinase have been made, and should allow tyrosinase cDNA clones to be isolated, either by providing an assay for the mRNA to be used in hybrid selection and translation of clones, or by allowing direct screening of cDNA expression libraries. Once a cDNA clone is isolated, mapping this

review gene with respect to the albino locus using Southern - " " blot hybridization will be straightforward.

Considerable interest is focused on the lethal albino deletions isolated by radiation mutagenesis at the MRC Radiobiology Unit, HarweU, UK and at Oakridge~XL In the Oakridge series over 3 600 000 animals were mutagenized and 119 c-locus mutations isolated. Of these, at least 34 were non-viable when homozygous and were studied further zs. A number were shown by enzymatic analysis to be deletions encompassing the mitochondrial malic enzyme locus (Mod-2) which maps at 1 cM from c. In two, the neuromuscular mutation shaker-1 had also been deleted. This deletion is over 4 cM and large enough to be cytologically detected. Complementation studies between the different mutations, coupled with an examination of the stage of lethality, permitted the mapping of a number of genes around albino. This map is shown in Fig. 4. The eight 'genes' detected are unlikely to be the only ones in this physically large region of the genome. The different groups of deletion end-points do not even necessarily define single genes. Furthermore, a gene which is located further from the albino locus than another which is lethal at an earlier point in development than this gene will not be seen by the analysis. The study provides a good genetic basis for free molecular dissection of a large chromosomal region once the c locus has been cloned.

Unstable genes Another allele of albino, chinchilla-mottled (c~), was

recovered after neutron-irradiation of the sperm of a chinchilla (c~/c ~) mouse t. Both homozygotes, c'/c M, and heterozygotes with chinchilla, c'/c ~h, have mottled fur. Some patches of hair are phenotypicallylike c~/c ~ whilst others are lighter and more like ~/c. Individuals differ widely in the amount of each colour, but the patches are finely grained and indistinct. A /rags-acting dominant gene, modifier of chinchilla mottled (Mcm-1), has been found t, which lightens the fur colour, but only when/rags to the c" allele. It is possible that c" functions as an unstable mutation, so that in some melanocyte clones it is 'on' and in others it is 'off, leading to the patchy phenotype. Mcm-I may then stabilize or change the rate of this instability. Mcm-1 is identifying a gene which is necessary for normal development, as homozygotes show a markedly reduced viability and are usually stunted in growth (Truslove and Deol, peps. commun.). Another modifier of chinchilla mottled, Mcm-2, which is not allelic to Mcm-1 has also been reported ~.

Several other genes show a similar type of phenotype to c ~ (reviewed in Ref. 1). Two alleles of agouti, agouti viable yellow (A~ and agouti mottled (a") produce a mottled coat; A" having agouti and yellow patches, and a" having non-agouti mixed with agouti. Three alleles of pink-eyed dilution, pink-eyed mottled 1 (p~), pink-eyed mottled 2 (p"¢) and pink- eyed unstable (p') have patches of fur which are diluted in colour (i.e. p/p) and others which are wild- type (P/-). The data for p" are strongest and indicate some form of reversion of the mutation to wild-type as the basis for the instability. Mostp'/p" orp~/p mice have a pale coat, with a few small patches of darker

r iews hairs. Occasionally an animal arises in which the patches are larger and more numerous (Fig. lb). These mice look very much like chimaeric animals and like chimaeras produce a number of wild-type offspring when mated to homozygous p ~ / p " or p/p mice (Truslove and Deol, pers. commun.). The wild- type gene in these offspring is stable and breeds true. It appears as though an event reverting t h e p " to wild- type occurs with a low probability. The event will tend to occur when the cell number is high, so producing small and few patches of wild-type tissue. Less fre- quently the event will occur earlier, when the cell number is low, giving rise to a substantial proportion of wild-type cells, in both the somatic and germinal tissues. The temptation is to ascribe this mutation to the action of a transposable element disrupting the wild-type p gene, to produce a lightened phenotype. The model supposes that the transposable element excises at variable times in development, giving rise to the high- or low-grade chimaeras observed.

S u m m a r y In conclusion then, the use of coat colour genes as

markers for mutagenesis studies has led to the identi- fication of many alleles at some loci. New mutations are appearing regularly in mouse stocks. Genes closely linked to coat colour genes have been found to have specific effects in development. The embryo- logical behaviour of melanocytes serves as a good model for the migration of cells during development and the action of genes on this migration. In the near future the techniques now available will provide molecular explanations for many interesting and informative genetic and developmental phenomena.

R e f e r e n c e s 1 Silvers, W. K. (1979) The Coat Colors of Mice, Springer

Verlag 2 Rawles, M. E. (1947) Origin of pigment cells from the neural

crest in the mouse embryo. Physiot Zool. 20, 248-266 3 Mint.z, B. (1987) Gene control of mammalian pigmentary

differentiation. I. Clonal origin of melanocytes. Proc. Natl Acad Sd. USA 58, 344-351 4 McLaren, A. (1976) Mammalian Chimaeras, pp. 52-59,

Cambridge University Press 5 McLaren, A. and Bowman, P. (1969) Mouse chimaeras

derived from fusion of embryos differing at nine genetic factors. Nature 224, 238-240 6 Silvers, W. K. and Russell, E. S. (1955) An experimental

approach to the action of genes at the agouti locus in the mouse. ]. Exp. Zoo/. 130, 199-220

7 Mayer, T. C. and Fishhane, J. L. (1972) Meso- derm--ectoderm interaction in the production of the agouti pig- mentation pattern in mice. Genet/cs 71,297-303

8 Poole, T. W. (1975) Dermal-epidermal interactions and the

T I G - December 1985

action of alleles at the agouti locus in the mouse. Dev. B/or 42, 203-210 9 Sakurai, T., Ochiai, H. and Takeuchi, T. (1975) Ultra-

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l. J. Jackson is at the MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, ~ N W 1 2HE, UK.