Heterokaryon Analysis of the Genetic Control of Pigment Synthesis in Chick Embryo Melanocytes

HETEROKARYON ANALYSIS OF THE GENETIC CONTROL OF PIGMENT SYNTHESIS IN CHICK EMBRYO MELANOCYTES

L. M. WILKINS,' J. A. BRUMBAUGH' AND I. W. MOORE" Cell Biology ond Genetics Section, School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68588

Manuscript received March 5, 1982 Rcviscd copy acccptcd July 24. 1982

ABSTRACT The gcnctic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, BI, e" and 1. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type wcrc fused to produce all tcn possihlc pair combinations of nondividing hotcrokaryons. Heterokaryons wcrc identified autoradiographically. (One partner in each pair was labclcd with "H- thymidinc.) Crosses produced comparable pairs of doublc hetcrozygotcs that were analyzed in vivo and in vitro. Heterokaryon pairs wcrc compared to their corrcsponding double hc:tcrozygotcs.--Some combinations showed complementation and produced wild-typc pigmcnt. Others showed noncomplemcntation having littltt or no pigmcnt. Double heterozygotes complemented each othcr except in thc cases involving tht: dominant mutant, I. Four hcterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-e' combinations failed to complement as heterokaryons hut did complc- mont as double heterozygotes. On the othcir hand, the I-c and I-€31 combinations complcmcnted as heterokaryons hut not as double heterozygotes. Based on thme diffcrcnces it is hypothesized that the pk and I loci are nuclearly rcstricted regulatory elements. Examples in the literature from othcr systcms arc cited to support such a hypothesis.

ERTEBRATE pigment synthesis is a complex process involving the con- V struction of an intracellular organelle, the 'melanosome. BACNARAet al. (1979) have reviewed the neural crest origin of pigment cells and their devel- opmental variations as expressed in vertebrates. Pigment granule synthesis involves two distinct cellular processes. One is concerned with the synthesis and appropriate packaging of the primary pigmentary enzyme, tryosinase, which involves Golgi-associated vesicles and cisternae. The second main process is the construction of the substructure of the pigment granule itself, the premelanosome. Premelanosome assembly occurs in dilations of the smooth endoplasmic reticulum as its components are sequestered. The Golgi-matured enzyme is then transported through coated vesicles to the enlarged membrane- bound vesicles containing the premelanosomal components where it becomes

' Prcsont aildrcss: Htmosliisis and Thrombosis Unit. Beth Isriicl Hospital, 330 Brookltnc Avcnuo. Boston, MA 112115. ' Corri:sp[)ndr:nr:[. shorild Iiv writ to: Dr. Iom BKIIMuAocH, School of Life Scimcr:s. Ilnivc~rsityof Nchraska. 1,inc:oln. NE 68588.. ' Priwnt addrcss: Di:pirtment of Biology, E;istc3m Coll~gv,S1. David's, PA 19087.

Genetics 102: 557-569 November. 1982 558 L. M. WILKINS. J. A. BRIJMBAlJCH AND J. W. MOORE activated and the pigment, melanin, is polymerized on the assembled premelanosomal matrix. This bipartite origin of pigment granule components has been demonstrated in studies by MAUL(1969), MAULand BR~JMBAUCH(1971), and BRUMBAIJGH,BOWERS and CHATTERJEE(1973). The somatic cell genetic analysis reported in this paper further explores the nature of pigment granule synthesis. Somatic cells differing at specific loci can be fused and complement a t'ion analyses and gene functions can be studied in the resulting nondividing heterokaryons (nuclei of different genotypes sharing a common cytoplasm). This has been applied to a number of human genetic anomalies. For example, several complementation groups have been found for xeroderma pigmentosum and for P-galactosidase mutants (BOOTSMAand GALJAARD 1979). In these studies. con- comitant examination of gene function in double heterozygous cells (monnkar- yons) could not be carried out. Heterokaryon studies of a similar nature have been applied to an investigation of pigment synthesis in chick embryo pigment cells by BRUMBAUCH,WILKINS and SCHALL(1978) and WILKINSand BRUMBAUCH (1979). This presentation is a survey of all combinations of pairs of five pigment mutants at five different, unlinked loci. Both heterokaryons and double heterozygotes were examined. The data reveal gene function through complementation or noncomplementation as measured in terms of normal pigment production. Recent investigations have demonstrated the existence of gene control elements in higher eukaryotes. For example, CHOVNICKet al. (1976) have demonstrated a cis acting control region coupled with the structural segment for the enzyme, xanthine dehydrogenase in Drosophila. In the mouse, complex loci have been postulated for various liver enzymes which contain both structural and contiguous contro1 elements. These same systems also have regulatory elements that are unliked or "distant" (PAIGEN1979; DANIEL,ABEnIN and LANGELAN1980). The agouti locus in the mouse, which controls the synthesis of black and yellow pigments, has been given a similar complex locus designation by SILVERS(1979) and GALBRAITH,WOLFF and BREWER(1980). Our results seem to indicate that unlinked regulatory elements are operating during the synthesis of pigment granules in the chick. Heterokaryon complementation analyses in Neurospora have indicated the existence of nonallelic regulatory elements whose site of action is restricted to the nucleus. CASEand GILES(1975) reported a slow complementing mutant involved with quinate metabolism. BURTONand METZENBERC(1972) showed that one of the loci involved in sulfur metabolism is a noncomplementing, nuclearly restricted regulator. GRIFFITHS(1976), through heterokaryon analysis, found the action of a supersuppressor mutant in Neurospora to be nuclearly restricted. The present study produced similar data with regard to the control of pigment synthesis and allows for similar interpretations, but with the advan- tage that gene dosage may be directly determined autoradiographically.

MATERIALS AND METHODS

Genetic stocks: Thc genoty.pt:s used in this study were obtained from stocks of domestic fowl maintained in thc School of Lifc Scicnces, [Jnivcrsity of Ncbraska. Thr: stocks wcrc: wild typc GENETIC CONTROL OF PIGMENTATION 559 (junglefowl), recessive white (c), pinkcye (pk), blue (Bf), dominant white (I) and recessive wheatcn (e'). Each of the mutant genotypes has a characteristic phenotype of rcduced (BI, I) or absent (c pk. e') pigment that is different from each of thc other mutant types. Thesc gnnotypcs havc: b(:t:n cxamined ultrastructurally and enzymatically. The major pigment-producing enzyme, tryosinasc (dopa oxidase), can be easily recognized at the ultrastructural level using an electron microscopic cytochemical test first reported by NQVIKOFF,ALBALA and BIEMPICA(1968) using melanomas and subsequently applied to chick material by MAULand BRIIMBAIJGH (1971). All genotypes have heen found to be tyrosinase positive, using this cytochemical tcst, except recessive white (c). Complete descriptions of mutant phenotypes, including their enzymatic properties as revealed ultrastructurally, have been prcviously rcportcd (BRIJMBAUGH1971; BRIIMBAIIGH and LEE 1975, 1976). These descriptions have also shown that cultured melanocytes closely resemble pigment cells of the same genotype when observed in vivo. Blue (El) is an incompletely dominant mutant that produces diluted pigmentation as a heterozygote (Bl/bl+) and almost white but flecked phenotype as a homozygote (BI/BI). Dominant white (I) is the other dominant mutation used in this study and its phenotype as a heterozygote (l/i+) is characterized by small black flecks that arc absent in the homozygote (I/I) (BRIJMBAIJGH1971). Cell culture and heterokaryon formation: The method of melanocyte cultura has previously been reported (BRUMBAUGH,WILKINS and SCHALL1978; WILKINSand BRIIMBAIIGH 1979). Trypsinized trunk regions from 72-hr embryos were plated out at moderate density (4 X IO5 cells/6O-mm dish) as monolayer cultures in a modified F-12 medium with 10% fetal bovine serum. After 3 days of apparent inactivity and dcplating, colonies of rapidly dividing melanoblasts appeared. In wild-type cultures, these cells began to form pigment and became frank melanocytes on day 5. Pigment cells accounted for 60-85% of the cell population. It was during the rapid proliferating period (3%-4% days) that the cultures chosen for 'H-thymidine labeling were exposed for 24 hr at 0.2 pCi/ml. Cell fusion procedure has also been previously described (BRUMBAIIGH,WILKINS and SCHALI. 1978; WILKINS and BRIIMBAIIGH 1979). Cells of each parental genotype were concurrently but separately cultured. One parental type was always labeled with "H-thymidine. On the 5th culture day the cells of each parental type were harvested, mixed and cocultured for 24 hr and then fused with UV-inactivated Sendai virus. All of the ten possible combinations were produced. Ninety to 95% of the radioactively treated parental melanocytes were labeled by tho "H-thymidine. Because both the blue and dominant white mutations did not completely eliminate pigment as visualizd with the light microscope, homokaryons were made by fusing pure cultures of these genotypes to determine dosage effects. Collection of heterokaryon data: Normally, cultures were terminated by fixation 48 hr after fusion. In one instance. the fusion of pinkeye (pk) with recessive wheaten (e "), some culturcs were held for 96 hr before fixation. Two different methods were used to determine the results of the fusions. First, subsequent to fixation but before autoradiographic processing and staining, each fused dish was scanned for heavily pigmented (wild-type appearing) cdls. Each of thcse cells was marked by a circle. A dissecting needle was used to scribe the circle on the outside of the bottom of the plastic culture dish. The number and genotype of the nuclei of these wild-type-pigmented cells was unknown at the time they were located. After the same dishes had been processed for autoradiography and stained, the nuclear makeup of the cells was recorded with particular regard to the number of nuclei and whether they were labthd or unlabeled. The second type of analysis was performed after autoradiographic development and staining. Each fused dish was systemically scanned, and all heterokaryons wcrc scorcd, recording the labeled and unlabeled nuclei and thc extent of pigmentation. The two methods provided a check on each other since pigmentation was the identifier in the first test and nuclear composition the identifier in the second test. During both types of scans homokaryons were also examined. Heterozygotes in vivo and in vitro: Crosses were made between each of the mutant types so that all of the ten possible combinations werc produced. The resulting chicks and adult fowl, which were heterozygous at two loci. werc classified with regard to degree and type of pigmentation. In three instances cells were cultured from doubly heterozygous embryos, namely Bl/bl+ C+/c. Bl/bI' Pk+/pk and I/i' Bl/bl'. The pigmentation of the cultured cells was then compared with that of chicks and adults of the same genotype. In one instance (l/i+ Bl/bl+), the cells were fuscd with each othr:r for direct comparison, as far as gene dosage was concerned, with comparable heterokaryons. The amount and type of pigment produced by thc crosses, cultures and homokaryons indicatcd 560 I.. M. WILKINS, I. A. RR[lMRAUCH AND 1. W. MOORE whcthw or not c:ompli:mc:ntiition hiitl oc:c:urrc:d. Thc linkagc: relationships 1ic:twc:c:n wc:h of thc fivc: loci wcrc iivnil;itilo from thc 1itf:r:itiirc. R ESIJ LTS Heterokaryons, as confirmed autoradiographically, fell into two groups with regard to melanin production. Some of the ten combinations produced normally pigmented cells, whereas others did not. The number of nuclei of each genotype in a given heterokaryon could be determined directly by counting' the number of labeled and unlabeled nuclei. Because approximately equal numbers of cells were cocultured and fused, the majority of heterokaryons contained one labeled and one unlabeled nucleus. Those with different nuclear ratios were noted, but no dosage effects were found. The presence of at least one labclcd and one unlabeled nucleus produced the characteristic phenotype for a given heterokaryon combination. Nuclear ratios of more than 3:l were rarely encountered. The presence of wild-type pigment in heterokaryons indicates complementation. This is not unlike complementation, as evidenced by restoration of normal function, in heterokaryons of fungi (FINCHAM,DAY and RADFORD1979) or human fibroblasts (BOOTSMAand GAL~AARD1979). The dcficient mutated function in one parental nucleus was supplied by the wild-type allele in the other parental nucleus and vice versa. Unpigmented or hypopigmented combinations indicated that complementation did not occur with certain mutant pairs. Complementation or noncomplementation was confirmed in each case by the two independent methods previously described. In Figure 1, a pigmented or

FI(;IJRE 1.-Blue:-rccc:ssivc whitc holcroknryon showing two Iti1)dcd nidci (Iiliick arrows) and on(! lahclod nuclt:ris (opm arrow) plus c:ytopliismic: pigmcnt grtinulos tlomonstriit ing c:omplmnc:ntii- lion. (X1400). GENETIC CONTROL OF I’IGMENTATION 561 complementing heterokaryon is shown containing two labeled nuclei and one unlabeled nucleus and abundant pigment granules. It is one of the blue (BI)- recessive white (c) combinations, (RI/BI C+/C+) + (bl+/bl+c/c). Figure 2 shows an unpigmented or noncomplementing heterokaryon of the pinkeye (pk)-recessive wheaten (e’) combination, (pk/pk E+/E’) + (Pk+/Pk+ e’/e’). It contains ono labeled and one unlabclcd nucleus, and there is no pigment in the cytoplasm. Table 1 displays the percentage of heavily pigmented cells marked before autoradiography that were confirmed as heterokaryons after autoradiography. Note that at least 74% of all the pigmented cells selected were heterokayrons in complementing combinations, whereas noncomplementing combinations had less than 15% heterokaryons. These numbers deviate from the theoretical 100 and O%, respectively, because blue (BI/BI) and dominant white (I/I) homozygotes produce some background pigment and can be misclassified. Also, some heterokaryons possibly became hybrid cells with only one nucleus. Confirma- tory karyotyping of putative hybrids was not possible because the differentiated melanocytes divided very slowly, if at all, after fusion. Table 2 displays the percentage of all heterokaryons, from systematically scanned dishes, that showed wild-type pigmentation. The same pairs indicate complementation and noncomplementation as those in Table 1. Complementing combinations possessed wild-type pigmentation in at least 36% of the heterokaryons observed, whereas noncomplementing combinations showed less than 4%)with wild-type pigmentation. One hundred percent of all heterokaryons

,r :-.: -” 562 L. M. WILKINS, 1. A. BRUMBAUGH AND J. W. MOORE

TABLE 1

Percentage of heavily pigmented cells confirmed (IS heterokaryons''

c- X pk - 99.1% X (106)b BI - 79.9% 7.3% X (134) (69) ey - 74.1% NP" 77.3% X (54) (269) I - 80.0% 14.0% 78.6% 13.7% X j439) (142) (140) (307) - C pk Bl ey 1 In each case two different experimtmts wt:re sampled. Heterokaryons contained at least one labeled and one unlabeled nucleus. Mutant and wild-type al1elt:s at each locus art: in separate nuclei, e.g., (Pk'/Pkt c/c) + (pk/pk C'/C+). I' Numbers in parentheses indicatc tht: numhnr of hcavily pigmcntcd cells evaluated for each combination.

I NP = no heavily pigmented cells observed; classified at 96 hr rather than tho usual 48 hr to ensure sufficient time for nuclear interaction.

TABLE 2

Percenluge of outorudiogruphicully determined heterokaryons" or homokoryonsh showing wild-type pigmentation'

c- NP/>d pk - <100'R' NP" BJ - 41 9% 3 5% 5 0%" (172)' (142) (161) e' - 43 4% NP' 41 1% NP" (106) (135) (185) I - 37 6% 13% 364% 3 0% 4 5%" (258) (152) (154) (166) (177) C pk BI cy r Contained at least one labeled and one unlabelrd nuclrus Mutant dnd wild-type allrlrs at rach locus are in srtparatr nucle~.e g , (Pk'IPk' c/c) + (pk/pk Ct/Cf) At least two nurlei of only onr grnotypr in a single cytoplasm ' In each case two differrnt experiments wPrr sampled " NP = no heavily pigmented cells observrd ' Not numerically evaluated but known to be less than 100% ' Numbers tn parrntheses indicate thr numhpr of hetrrokaryons or homokaryons evaluated for each combination Classified at 96 hr rather than thr usual 48 hr to rnsure sufficient time for nurlear intrraction would be expected, theoretically, to produce pigment in the complementing pairs. This is not achieved, however, possibly because of individual variations in the stages of development of the cells within d culture with regard to pigment synthesis. Again, the dominant white (I) and blue (BI) mutants produce some pigment which accounts for the 1-4% "background" of pigmented cells observed in some noncomplementing pairs. This "background" level was confirmed by producing blue (SI)and dominant white (I) homokaryons. Five and 4.5% of blue (BI) and dominant white (I) homokaryons, respectively, were classified as possessing wild-type pigment (Table 2). GENETIC CONTROL OF PIGMENTATION 563

The results of the heterokaryon complementation tests are summarized in Figure 3. Three generalized statements can be made about the results. First, all tested mutations readily complemented with recessive white (c).Second, the pinkeye (pk) mutation is singled out by the fact that it does not complement with the three remaining mutations, blue (SI), recessive wheaten (e') and dominant white (I). Third, among the three mutations that do not complement with pinkeye, there is the ability to complement in two of the three cases. Only the dominant white (I)-recessive wheaten (e') combination is unable to produce wild-type pigment. The results of the double heterozygote complementation tests are also summarized in Figure 3. Here, interpretation is complicated by the fact that blue (BJ) is an incompletely dominant mutation and dominant white (I) a completely dominant mutation. According to FINCHAM(1966), the production of gene product less abnormal than either mutant parent in the heterozygotes indicates that each normal allele can correct at least partially the defect in the other. Blue heterozygotes (Bl/bJ') clearly produce significant pigment even when heterozygous at other loci. Thus, double heterozygote combinations involving blue (BJ) show incomplete complementation (i, Figure 3). Dominant white (I) combinations are difficult to interpret because of the somatic instability of the mutation. Heterozygotes are easily detectable from homozygotes in vivo because of the small black flecks that occur on the otherwise white background. The white background, however, does not differ between the homozygotes and the heterozygotes. Thus, we have treated dominant white as a completely dominant

FIGVRE3.-Summary and comparison of pigment production in heterokaryon pair combinations and double heterozygote pair combinations. Heterokaryon results arc indicated in the upper left of cach box, whercas double heterozygote results are indicated in the lower right of cach box. + = wild-typc pigment production or complementation: I = partial pigment production or inLomp1t:te complementation; - = little or no pigment production or noncomplemcntation. 564 1,. M. WlLKINS. 1. A. BRUMBAllGH AND 1. W. MOORE mutation and have indicated that it does not complement with any of the other mutations in the double heterozygous state (-, Figure 3). In summary, all combinations at least partially complement as double heterozygotes except those involving dominant white. Comparisons of the results of heterokaryon complementation tests with double heterozygote complementation tests reveal four noncoordinate cases. In two instances involving pinkeye (pk) the double heterozygotes complemented, whereas the heterokaryons did not. These were the pinkeye (pk)-blue (BI) and pinkeye (pk)-recessive wheaten (e ’) combinations (Figure 3). In the other two cases, involving dominant white (I), the double heterozygotes did not complement, whereas the heterokaryons did. These were the dominant white (I)- recessive white (c) and dominant white (I)-blue (BI) combinations (Figure 3). Heterokaryons between dominant white (I)and blue (BJ) readily complemented, (I/I bI+/bJ+) + (i+/i+ BI/B1). Cells heterozygous for these mutations (I/i+ bl’/BI) were cultured and fused with themselves, and homokaryons were examined for pigment production, (l/i+ bl+/BI) + (l/i+ bl+/Bl). These double heterozygotes, even as homokaryons, did not produce wild-type pigment. Linkage tests reveal that none of the mutants are alleles since they are not linked to each other (WILKINSand BRUMBAUCH1979; WARREN1949).

DISCUSSION In this study complementation in heterokaryons could be explained in several ways. Since two different genotypes grow together in the same culture dish, cellular crossfeeding of diffusible products could be the reason for restored normal function. Complementation in mixed hut unfused cultures does not occur, thus eliminating this possibility (BRUMBAUGH,WILKINS and SCHALL1978). A second explanation, applicable only to heterokaryons, would suggest that the act of fusion itself brings together previously synthesized cytoplasmic components that are then free to interact and assemble in the common cytoplasm of the heterokaryon to form functional products. This has been demonstrated for certain types of heterokaryon complementations involving human P-galactosidase-deficient variants (DE WIT-VERBEEK,HOOCEVEEN and GAL~AARD 1978). This explanation does not appply, however, to double heterozygotes that do not start out with previously synthesized gene products. Complementation is defined by FINCHAM,DAY and RADFORD(1979) as follows (p. 74): “If the chromosome sets of two defective mutants are brought together into the same cell, whethcr by heterokaryon formation or by formation of a diploid nucleus, a normal phenotype will result if each mutant can supply what is deficient in the other.. . .” The top portion of Figure 4 diagrammatically represents this explanation of complementation occurring in a heterokaryon between recessive wheaten (e’) and recessive white (c).The synthesis of normal pigment granule components by the normal alleles in each nucleus and their interaction and assembly in the common cytoplasm are presented. Since the five loci studied are unlinked and, therefore, not alleles, they should all complement each other. When the double heterozygote tests are examined, (Figure 3) it is clear that four complementation groups are represented by the GENETIC CONTROL OF PIGMENTATION 565

~ ~~~ FIGURE4.-Top: Model depicting complementation between recessive white (c) and recessive wheaten (e-").Normal C and E products are produced and combined in the common cytoplasm and wild-type pigmentation results. Bottom: Model depicting noncumplemcntation t)ctwt:en pinkeyc (pk) and recessive wheaten (e")using the Pk locus as a putative nuclearly rcstrictcd regulator. The left nucleus produces only mutant (el) gene product, whereas the right nucleus, hccausc of tha mutant pk allele, docs not produce E gcno product. The normal Pk product from tho Icft nuclcus is prevented from exerting normal control over the normal E locus in tho right nuclcus by thc nuclcar membranes and/or cytoplasm. .--f = normal gene product; + = abnormal gcnc product; A( = incffective or blocked gene product. four mutations: recessive white (c), pinkeye (pk), blue (BI) and recessive wheaten (eY).The cases of noncomplementation involve dominant white (I) and are probably associated with its dominance. 566 L. M. WILKINS. I. A. BRLJMBAUGH AND J. W. MOORE Two noncomplementing heterokaryon combinations occurred, whose corresponding double heterozygotes did complement, namely, blue (El)-pinkeye (pk) and recessive wheaten (e”)-pinkeye (pk).This difference in response between heterokaryons and diploids is not unprecedented in fungal genetics (FINCHAM, DAYand RADFORD 1979). Explanations for such differences involve the incorn- plete mixing of the cytoplasm and/or widely varying nuclear ratios of the two different genotypes involved in heterokaryon formation. In this study the number of nuclei of each contributing genotype in heterokaryons can be read directly by autoradiography. Since complementation occurs in a majority of combinations, cytoplasmic mixing does occur. The fact that a given mutant will complement with another mutant in a heterokaryon combination but not with a third mutant shows that it is not an inherent methodological problem that accounts for the lack of wild-type pigment in the noncomplementing combinations. FINCHAM,DAY and RADFORD(1979) continue their explanation referring to noncomplementation as follows (p. 74): “. . ., but a mutant phenotype, probably resembling the less extreme of the two mutants where they are different would be obtained if both chromosome sets are deficient in the same function” (italics added by the authors of this paper for emphasis). Therefore, noncomplementing heterokaryon combinations in this study apparently involve mutants affecting the same pigmentary function. Plausible explanations for this nonallelic noncomplementation in heterokaryons have been provided by three studies in Neurospora. CASEand GILES(1975) found that certain heterokaryon combinations involving mutants of quinate metabolism produced very slow complementing heterokaryons. They suggested that slow complementation was due to the fact that the gene function under study acted primarily within its own nucleus and was supplied only slowly to the other nucleus of the heterokaryon. BURTONand METZENBERC(1972) showed that the scon‘ mutation, involved in sulfur nutrition, was limited in its effect to its own nucleus. GRIFFITHS(1976) showed that supersuppressor (SSU) gene products were also nuclearly restricted in heterokaryons. In all three instances the nuclearly restricted mutations appeared to be regulatory elements. Since the pinkeye (pk) mutation is involved in both cases of noncomplementation in heterokaryons, it too might be a regulatory element limited or partially restricted in its activity to its own nucleus. It can also be suggested that the noncomplementing partners with pinkeye (EJ; e’) are mutants involved with the same function as pinkeye. Attempts were made to allow extra time for nuclear interaction in the pinkeye (pk)-recessive wheaten (e’) combination. Instead of evaluating heterokaryons 48 hr after fusion, they were evaluated at 96 hr. This did not permit these heterokaryons to produce pigment. The bottom portion of Figure 4 diagrams a heterokaryon between pinkeye (pk) and recessive wheaten (e’) showing the noncomplementation resulting from the hypothetical nuclearly restricted action of the pinkeye (Pk) locus. In addition to the two cases in which heterokaryons failed to complement but the double heterozygotes did, two converse cases occurred. The dominant white (1)-recessive white (c) and dominant white ([)-blue (El) combinations failed to GENETIC CONTROL OF PIGMENTATION 567 complement as double heterozygotes but readily complemented as heterokaryons (Figure 3). Here again, the disparity between heterokaryons and diploids could be explained on the basis of the nuclear restriction of a putative regulatory element. If the mutant, dominant white (I), has an inhibitory action upon some aspect of pigment synthesis by affecting another locus, and if its action is restricted to the nucleus, then it would be expected to cause mutant function in ail double heterozygotes (which it does; Figure 3), since the mutant, I, is in the same nucleus as the locus it affects. Thus, it acts as a dominant mutation. In a heterokaryon, however, the effect of dominant white would be restricted to its own nucleus and could not alter pigment synthesis in the other nucleus. If the mutant in the other nucleus was not involved with the same pigmentary function as I, then complementation would result. Thus, it can be suggested that recessive white (c) and blue (BJ) affect different pigment cell functions from dominant white (I). An explicit check of dosage in the case of the dominant white (I)-blue (BI) combination was undertaken. Double heterozygotes between blue (B1) and dominant white (I) were fused with themselves to produce binucleated homokaryons that could then be compared with binucleated heterokaryons. Gene dosage and number of nuclei would be the same in each case, but the arrangement of mutant and normal alleles would be altered. They could be symbolized as follows: binucleated homokaryon, ( I/it bJ+/BJ) + (I/i+ bl+/Bl); binucleated heterokaryon, (I/I bl+/bl+) + (i+/i+BJ/Bl). The homokaryons produced little or no pigment, whereas the heterokaryons produced copious amounts at the wild- type level. Thus, the difference between heterozygotes and heterokaryons is not due to dosage effects but to gene arrangement in the nuclei. BAGNARAet al. (1979) have summarized the morphological and biochemical data with regard to pigment granule synthesis. They conclude that at least two major types of components are involved. The enzyme, tyrosinase, is primarily responsible for the oxidation of substrate (tyrosine and/or dihydroxyphenylal- anine) to melanin precursors. The lattice-like pigment granule matrix or premelanosome is less well defined but seems to provide sites for melanin depo- sition. These two major components are brought together in the cell and, after tyrosinase activation, melanin polymer is laid down upon the matrix and an opaque pigment granule develops in genetically normal types. Thus, pigment granules can be described as consisting of tyrosinase and nontyrosinase or premelanosomal components. Based on heterokaryon complementation data alone these two major groups become apparent. All mutations complement with recessive white (c)which is the only enzymatically negative mutation of the five used. The Pk locus presents itself as a major gene involved in nontyrosinase component (premelanosome) synthesis or assembly since none of the other tyrosinase-positive mutations (BJ, e’, I) can complement with it. Within the Pk group there is complementation in two of the three combinations. This suggests that at least two products are involved in the nontyrosinase aspects of pigment granule synthesis. Based on the differences between heterokaryon and diploid heterozygote results, it seems likely that the I locus is a regulator lower in the genetic hierarchy than the Pk 568 L, M. WILKINS, J. A. BRUMBAUGH AND J. W. MOORE locus since it apparently controls just one of the two putative nontyrosinase components (E locus). Thus, two putative regulatory elements have been identified by heterokaryon complementation analyses of five pigment mutants in the fowl.

This study was supported by grant GM 18969 from the National Institutr: of Genwal Medical Sciences.

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