Differentiation Of Melanosomes Research Articles

MAPK Pathway Inhibitors Sensitize BRAF-Mutant Melanoma to an Antibody-Drug Conjugate Targeting GPNMB.

To determine if BRAF and/or MEK inhibitor-induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB. The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth. A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor-treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone. The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088-98. ©2016 AACR.

Abstract 296: Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma

Abstract Background: The duration of survival benefit from MAPKi in B-raf mutant melanoma (MEL) is limited by the near universal development of resistance (RES). Recently, MiTF, a transcription factor and master regulator of melanocyte differentiation - was identified as a hub of MAPKi RES. Given its dual role as both a tumor promoter and inducer of MEL senescence, MiTF is a problematic therapeutic target. Pigmentation is a MiTF-dependent feature of MELs correlates with poor survival. We have interrogated the MAPKi/MiTF-dependent regulation of a panel of MITF-regulated melanosomal differentiation antigens (MDAs) for their potential as therapeutic targets to overcome MAPKi RES. Methods: The TCGA MEL gene expression data set was interrogated. B-raf mutant MEL cells were exposed to dabrafenib (D), or trametinib (T). qRT-PCR, Immunoblot or FACS were used to assess MiTF and MDA expression. Serial biopsy tissue and serum samples from MEL patients taken before treatment, during treatment, and at progression with MAPKi therapy were obtained, and analyzed by IHC and qRT-PCR, RNAseq, and ELISA. Transient siRNA-mediated knockdown was used to assess the requirement for MiTF, in MAPKi-mediated induction of MDAs, including GPNMB. To assess the interaction between MAPKi and CDX011 (C), a GPNMB- targeted antibody drug conjugate, mice bearing A375 and WM2664 xenografts were treated with T or DT + C in combination. Results: MiTF is strongly correlated with MDAs, including GPNMB in gene-expression datasets derived from human MEL tumors. A MiTF driven MDA-gene-signature correlates with poor survival among 291 melanoma patients. MAPKi treatment increased MiTF and MDAs in MEL cells, and induced pigmentation in vivo. This phenotype was reversible as MEL acquired RES to MAPKi. Knockdown of MiTF leads to abrogation of MAPKi-mediated GPNMB induction. GPNMB promotes MEL invasion in vitro. MiTF and MDA were elevated in tumors and serum from patients receiving MAPKi treatment and returned to baseline in tumors that acquired RES. The addition of C to either T or DT led to, decreased pigmentation, prolonged tumor regression and inhibited the development RES to T or DT treatment in vivo. Tumors that acquired RES to DT displayed increased c-Met expression and MAPK activation; tumors treated with DT+C failed to develop this phenotype. Conclusions: MAPKi induce MDA in a MiTF-dependent manner in MEL. This process leads to melanocytic differentiation and increased pigmentation, which is subsequently reversed as a tumor acquires RES to MAPKi. This demonstrates the plasticity of MEL in response to MAPKi. The pigmented phenotype is less proliferative but more invasive which is in part mediated by GPNMB. This transient phenotype may allow tumors to acquire characteristics (ie. c-Met upregulation and MAPK re-activation) which contribute to MAPKi RES. Targeting a pro-invasive MDA, GPNMB with CDX-011 subverts this plasticity and impairs the development of MAPKi RES. Citation Format: April A.N. Rose, Matthew G. Annis, Dennie T. Frederick, Zhifeng Dong, Lawrence Kwong, Tibor Keller, Thomas Hawthorne, Ian R. Watson, Keith T. Flaherty, Peter M. Siegel. Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 296.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Induction of autoantibodies to syngeneic prostate-specific membrane antigen by xenogeneic vaccination.

Prostate-specific membrane antigen (PSMA) is a prototypical differentiation antigen expressed on normal and neoplastic prostate epithelial cells, and on the neovasculature of many solid tumors. Monoclonal antibodies specific for PSMA are in development as therapeutic agents. Methodologies to actively immunize against PSMA may be limited by immunologic ignorance and/or tolerance that restrict the response to self-antigens. Our studies have previously shown that xenogeneic immunization with DNA vaccines encoding melanosomal differentiation antigens induces immunity in a mouse melanoma model. Here we apply this approach to PSMA to establish proof of principle in a mouse model. Immunization with xenogeneic human PSMA protein or DNA induced antibodies to both human and mouse PSMA in mice. Monoclonal antibodies specific for mouse PSMA were generated to analyze antibody isotypes and specificity for native and denatured PSMA at the clonal level. Most antibodies recognized denatured PSMA, but C57BL/6 mice immunized with xenogeneic PSMA DNA followed by a final boost with xenogeneic PSMA protein yielded autoantibodies that reacted with native folded mouse PSMA. Monoclonal antibodies were used to confirm the expression of PSMA protein in normal mouse kidney. These results establish the basis for clinical trials to test PSMA DNA vaccines in patients with solid tumors that either express PSMA directly or that depend on normal endothelial cells expressing PSMA for their continued growth.

Xenogeneic Human Tyrosinase DNA Vaccination Induces Specific Immune Response in Dogs with Advanced Malignant Melanoma

Introduction: Xenogeneic melanosomal differentiation antigens, delivered in the form of a plasmid DNA vaccine, can overcome host immune ignorance/tolerance in preclinical animals to melanoma by: 1) generating humoral and cytotoxic T cell responses and 2) inducing protection from tumor challenge. Initial trials of human tyrosinase (huTyr) DNA vaccination of dogs with advanced malignant melanoma (Bergman et al, 2003) demonstrated safety and prolongation of survival with this therapeutic modality. We investigated antigen‐specific immunity in dogs receiving huTyr DNA vaccination.Methods: Three cohorts of three dogs each with advanced (WHO stage II‐IV) canine malignant melanoma (CMM) received four biweekly IM injections (dose levels 100, 500, or 1,500 ug, respectively/vaccination) of huTyr plasmid DNA via the Biojector2000 jet delivery device. Sera samples were taken before and after vaccination to detect specific antibody formation to huTyr by Enzyme‐Linked ImmunoSorbent Assays (ELISAs) and flow‐cytometry.Results: Three dogs have measurable, 2 to 4 fold huTyr‐specific antibody titers. Preliminary studies by flow‐cytometry have confirmed antibody response to huTyr by positive binding to endogenous human tyrosinase in SK‐Mel188 cells using post‐vaccinate serum.Conclusions: Xenogeneic (huTyr) DNA vaccination generates antigen‐specific humoral responses, which may partially explain the previously reported clinical efficacy and anti‐tumor responses. Ongoing studies include: 1) a comprehensive analysis by flow‐cytometry to detect huTyr‐specific antibodies and 2) a quantitative measure of potential cytotoxic T‐cell responses in these dogs by the DNA vaccination.

On the role of melanoma-specific CD8+ T-cell immunity in disease progression of advanced-stage melanoma patients.

Cytotoxic T-cell immunity directed against melanosomal differentiation antigens is arguably the best-studied and most prevalent form of tumor-specific T-cell immunity in humans. Despite this, the role of T-cell responses directed against melanosomal antigens in disease progression has not been elucidated. To address this issue, we have related the presence of circulating melanoma-specific T cells with disease progression and survival in a large cohort of patients with advanced-stage melanoma who had not received prior treatment. In 42 (68%) of 62 patients, melanoma-specific T cells were detected, sometimes in surprisingly large numbers. Disease progression during treatment was more frequent in patients with circulating melanoma-specific T cells, and mean survival of patients with circulating melanoma-specific T cells was equal to the survival of patients without melanoma-specific T cells. These data suggest that the induction of melanosomal differentiation antigen-specific T-cell reactivity in advanced stage melanoma is a late event most likely due to antigen load and spreading and is not accompanied by a clinically significant antitumor effect. These melanoma-specific T cells may be functionally distinct from T cells raised during spontaneous regression or up vaccination.

Antimelanogenic activity of hydrocoumarins in cultured normal human melanocytes by stimulating intracellular glutathione synthesis.

The antimelanogenic activity of six hydrocoumarins and alpha-tocopherol (alpha-Toc) in normal human melanocytes was evaluated in both cell culture systems and cell homogenates. The inhibitory effects of hydrocoumarins depended upon their substituent groups. alpha-Toc and some of the hydrocoumarins inhibited melanogenesis in cultured normal human melanocytes, although they did not influence melanin synthesis in enzyme solution prepared as cell homogenates. In addition, alpha-Toc and the hydrocoumarins stimulated intracellular glutathione (GSH) synthesis. In particular, 7-allyl-6-hydroxy-4,4,5,8-tetramethylhydrocoumarin strongly inhibited melanogenesis and intracellular GSH synthesis in normal human melanocytes, more so than alpha-Toc. Furthermore, hydrocoumarins exhibited higher scavenging and quenching activities against with tert-butyl peroxyl radicals and singlet oxygen species. These results suggest that 7-allyl-6-hydroxy-4,4,5,8-tetramethyl hydrocoumarin would be useful as an antimelanogenic agent for the prevention or improvement of skin pigmentation induced by reactive oxygen compounds and free radicals, and may inhibit melanogenesis, including tyrosinase transfer and melanosome differentiation, by interrupting melanization by increasing the intracellular GSH content.

Evolution of ptygmatic folds in migmatites from the type area (S. Finland)

It is proposed that ptygmatic folds in migmatites form during leucosome–melanosome differentiation. Volume loss (by depletion of quartz and feldspar) takes place in the interlimb domains of folds, leaving a residual melanosome. Buckling leucosomes increase in volume due to this process and are protected from substantial thinning. Concurrent with tectonic segregation, albeit at a lower rate, the leucosome expands by replacing the melanosome. This process is induced by fluid activity at the leucosome boundary, as suggested by the occurrence of secondary offshoot veinlets. Segregation and growth causes the most typical aspect of ptygmatic folds: preservation of an overall buckle shape during the stage when the folds become isoclinal and devoid of interlimb matrix.

Effects of Exogenous MSH on the Transformation from Phaeo- to Eumelanogenesis Within C57BL/6J-Ay/a Hairbulb Melanocytes

The extent to which alpha melanocyte-stimulating hormone (MSH) is a true in vivo regulator of melanogenesis in mice is unknown. The objective of this study was to determine if MSH-induced eumelanogenesis in hairbulb melanocytes of yellow (Ay/a) mice mimics the natural program of eumelanogenesis occurring in genetically black (a/a) hairbulb melanocytes. We conducted quantitative transmission electron microscopy on melanosome differentiation within MSH-treated regenerating 9-d hairbulbs of Ay/a and a/a mice. Results of exogenous alpha-MSH injections (5 d at 0.15 mM MSH) showed that the striking visual darkening of hair was accompanied by an incomplete transformation of phaeo- to eumelanogenesis. Ontogenetic data on developmental stages I-IV of 3678 melanosomes based on geometric considerations (length, width, shape, and area) showed that MSH did not induce a complete transformation from spherical phaeomelanosomes to elliptical eumelanosomes. Also, observations on the number of vesiculoglobular bodies and matrix organization reveled that MSH-treated Ay/a melanosomes retained distinct features of phaeomelanogenesis even after 5 d of MSH treatment. Thus, MSH induced a partial but incomplete pattern of eumelanogenesis in regenerating hairbulb melanocytes of Ay/a mice. The continued investigation of the dynamics of melanin synthesis in MSH-induced Ay/a mice melanocytes possessing "mosaic" melanosomes could be productive in understanding fundamental relationships between tyrosinase activity, matrix function, matrix structure, and regulation of melanin (phaeo- and/or eumelanin) synthesis.

Differentiation of Hairbulb Pigment Cell Melanosomes in Compound Agouti and Albino Locus Mouse Mutants (Ay, a, c2J; C57BL/6J)

Our objective was to determine using electron microscopy how nonagouti (a), lethal yellow (Ay), and albino (c2J) genes affect the program of mouse hairbulb melanosome differentiation; 1,921 hairbulb melanosomes from four genotypes (a/a C/C = B,Ay/a C/C = Y, a/a c2J/c2J = BA, and Ay/a c2J/c2J = YA) were scored for developmental stage, length, and width. Qualitative and quantitative electron microscopy revealed the following. An albino locus-induced diminution of melanosome size suggests that the albino locus is involved in structural features of melanosomes not directly related to the synthesis and deployment of tyrosinase. Ratio data on melanosome length-to-width confirm that the agouti locus determines melanosome shape, either spherical or elliptical; melanization is not required for melanosomes to achieve their agouti-locus-determined shapes. YA (Ay/a c2J/c2J) melanosomes, characterized by poorly organized matrices, absence of active tyrosinase, unusually large membrane invaginations, and significantly smaller dimensions than those of BA (a/a c2J/c2J), showed additive effects of both Ay and c2J alleles. These data suggest that the albino locus plays a structural as well as functional (tyrosinase) role in the differentiation of mouse hairbulb melanosomes. The agouti locus, even in the absence of melanization, directs melanosome shape either via synthesis and deployment of agouti-locus-encoded matrix proteins or by other structural factors. The additive effects of Ay and c2J alleles in compound YA mutants document the importance of specific interactions both functional and structural between agouti and albino loci.

Analysis of Initial Melanogenesis Including Tyrosinase Transfer and Melanosome Differentiation Though Interrupted Melanization by Glutathione

Because glycosylation-dependent melanization inhibition induced in cultured B-16 melanoma cells by glucosamine is reversible, producing synchronized initiation of melanogenesis after its removal, we have analyzed the possible dynamics of initial melanogenesis through their interruption by glutathione. The addition of glutathione at 0.2% concentration to the theophylline-stimulated recovery process completely interrupts the initiation of melanization for at least 72 h. At the electron microscopic level, theophylline-treated cells have many vacuolar melanosomes with distinct pigmentation which contain some vesicles (64% of total premelanosomes) or amorphous, filamentous, or granular materials within the interior which are suggestive of pheomelanotic melanosomes. The addition of glutathione induces a complete absence of melanization in the premelanosomes, within which a filamentous interior with periodicity is generally re-formed with almost complete disappearance of internal vesicles, providing dramatic changes to the size and shape characteristic of eumelanotic melanosome. Electron microscopic dopa reaction of glutathione-treated cells shows a predominant localization of tyrosinase activity in the Golgi-associated endoplasmic reticulum-lysosome and coated vesicles, but not in premelanosomes, in contrast to their dispersed distribution in all melanogenic organelles in the theophylline-treated control, suggesting a lack of tyrosinase translocation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of tyrosinase in the large granule fraction shows that in analogy with electron microscopic observations, glutathione blocks the reappearance of membrane-bound T3 tyrosinase which occurs in the theophylline-treated control during the recovery process, whereas the dynamics of T1 tyrosinase is almost the same as that of the control. These findings suggest that glutathione provides a new situation of interrupted melanogenesis in which melanization cannot proceed despite complete formation of melanosome matrix structure and a lack of inhibition of cellular metabolisms including protein glycosylation.

Analysis of melanosomes from cattle eyes by pyrolysis—gas chromatography

The application of Curie-point pyrolysis—gas chromatography—mass spectrometry to the differentiation of melanosomes isolated from the retinal pigment epithelium, ciliary body and iris of cattle eyes, with special attention to the preparation and the characterization of their melanin part, was investigated. Computer data analysis and evaluation of the pyrograms obtained was used to display variations between the pyrograms of melanosomes and their components represented by sets of peak heights for comparative studies. The mechanisms of formation of the pyrolytic products and the usefulness of the proposed procedure based on pattern recognition in view of biochemical processes in melanosomes are discussed.

Characterization of Structural Properties for Morphologic Differentiation of Melanosomes. III. Free and Protein-bound Dopa and 5-S-Cysteinyldopa in B16 and Harding-Passey Melanomas

This study analyzed the free and protein-bound forms of dopa, 5-S-cysteinyldopa (5-S-CD), and 5-S-glutathionedopa (5-S-GD) in B16 and Harding-Passey (HP) mouse melanomas to investigate the role of these catechols for melanogenesis and melanosome morphogenesis, inasmuch as these tumors produce melanosomes different in color and ultrastructure, i.e., eumelanosome type in B16 and pheomelanosome type in HP. Between B16 and HP mouse melanomas, however, we found (a) no significant difference in the level of free dopa and 5-S-CD in melanosomes and tumors, although the levels of these catechols reflected well the type of melanogenesis in control hair of normal mice, (b) a significant difference in free 5-S-GD level, which might, in part, reflect the observed difference in melanogenesis, and (c) no apparent difference in the level of bound dopa and 5-S-CD in either melanosomes or tumors. Thus, the striking difference in the color of melanosomes between B16 and HP melanomas seems to be related primarily to the content--not the type--of melanin pigments.

Characterization of Structural Properties for Morphological Differentiation of Melanosomes: II. Electron Microscopic and SDS-PAGE Comparison of Melanosomal Matrix Proteins in B16 and Harding Passey Melanomas

This study compared the qualitative and quantitative differences of the melanosomal matrix proteins between the ellipsoidal-lamellar melanosomes of B16 melanoma and the spherical-granular melanosomes of Harding Passey (HP) melanoma by electron microscopy and SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The purity of the melanosomes at each step of the sample preparations was assayed by % distribution of cytoplasmic marker enzymes and proteins. Characterization of the melanosomal matrix was carried out in material purified by the 2 steps of sucrose density gradient ultracentrifugation and membrane dissociation with BRIJ-35. Eighty-three percent of the matrix proteins were solubilized from both B16 and HP by guanidine HCl (GH), which also caused marked ultrastructural changes in different ways. The inner matrix of HP completely disintegrated into fine grains whereas the matrix of B16 showed a great loss in electron density and was degraded, though its basic framework remained unchanged. Under SDS-PAGE, the melanosomal matrix was dissociated into 14 polypeptide bands with respect to size and charge density, 10 of which (63-85% of total proteins solubilized by GH) were common to both B16 and HP. A significant quantitative difference was noted in the relative amounts of these common proteins. The remaining 4 were unique in molecular weight to each form of melanosome though their relative amounts were low. It is suggested that these chemical differences, i.e., quantitative and qualitative, of the matrix proteins are responsible for the ultrastructural differentiation of melanosomes.

Characterization of Structural Properties for Morphological Differentiation of Melanosomes: I. Purification of Tyrosinase by Tyrosine Affinity Chromatography and its Characterization in B16 and Harding Passey Melanomas

To characterize how structural properties are related to the morphological differentiation of melanosomes, two forms of melanosomes, commonly seen in mammals, were isolated from B16 and Harding Passey (HP) mouse melanomas. From these morphologically different melanosomes, tyrosinase was solubilized by BRIJ-35 and purified by affinity chromatographies substituted with tyrosinase substrates. We found that tyrosine ethyl ester (TEE) and dopa are effective and specific in retaining tyrosinase as an affinity media and that enzyme retrieval with approximately 100% recovery is possible by a substrate, TEE, or a competitive inhibitor, N-acetyl L-tyrosine. The purified tyrosinase of B16 and HP melanosomes possessed a common antigenic site and revealed little difference in size and the Mikaelis constant for dopa utilization. It is likely that tyrosinase is involved in melanosome morphogenesis only through melanization, and that it is not directly related to the architecture, i.e., lamellar and granular patterns, of the inner matrix.

Comparison of Eumelanogenesis and Pheomelanogenesis in Retinal and Follicular Melanocytes; Role of Vesculo-globular Bodies in Melanosome Differentiation

Vesiculo-globular bodies, 40 nm in diameter, are present in melanosomes. The mode of their involvement in melanosomal differentiation was studied by ultrastructural comparison of eu- and pheomelanogenesis occurring in retinal and follicular melanocytes. We found that the number and distribution of these bodies differ significantly with types of melanogenesis and tissues. They were not affected by physical stimuli nor by embryonic origin of melanocytes. The earliest form of melanosomes is identical in eu- and pheomelanogenesis. The vesiculo-globular bodies are involved in organization of melanosomal constituents. In eumelanogenesis, they are more numerous in feather than in retina and hair. They are least numerous in white hair and pink eyes where melanization is blocked. During melanosomal development, they become associated with melanosomal inner lamellae and their outer surface becomes melanized, but their core is hardly melanized, thus leaving small vesicular structures. In pheomelanogenesis, their number is almost equal in feather and hair. Lamellae are not formed, but these bodies fuse with each other to form an amorphous matrix on complete differentiation of melanosomes.

Ultrastructural analysis of gene interaction and melanosome differentiation in the retinal pigment cells of the albino goldfish.

The genotype of albino goldfish is represented by pp,cc, and the non-albino fish is PP,CC. The P gene begins to control the melanosome formation of retinal pigment cells at St-21; and the C, which is epistatic to P and p, governs melanosome formation in the dermal melanophores and in retinal pigment cells after St-22. Then in the pp,CC fish, between St-21 and St-22, melanosomes of the pigmented retina are albino in type, but after St-22 they become non-albino in type. From ultrastructural observations on the melanosomes of these late-melanizing fish, it is apparent that the imcompletely melanized albino pigment granules can differentiate into normal melanosomes under the control of the C gene. The processes of melanosome maturation in the late-melanizing goldfish are evident from these observations.

Genetic and developmental analysis of some new color mutants in the goldfish, Carassius auratus.

The genotypes of three color mutants in goldfish: a depigmentation character of larval melanophores, albinism and a recessive-transparent character, were analyzed by crossing experiments. The depigmentation character in the common goldfish is controlled by two dominant multiple genes, Dp1 and Dp2, and only fish with double recessive alleles dp1dp1 dp2dp2 can retain larval melanophores throughout life. Albinism is also controlled by double autosomal genes, p and c. The genotype of an albino fish is represented by PP CC; the non-albino fish is PP CC. Fish with either a pp CC or pp Cc genotype are albino when scored at the time of melanosome differentiation in the pigment retina, but after the time of skin melanophore diffrentiation, they change to the nonalbino type under the control of the C gene. The recessive-transparent character is controlled by a single autosomal gene, g. The mechanisms of gene expression of these characters were proposed as a result of observation and/or experimental data on the differentiation processes of their phenotypes, and the genotypes of these color mutant goldfish were considered in relation to the "gene duplication hypothesis in the Cyprinidae."