Coat color dilution in mice because of inactivation of the melanoma antigen MART-1

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.

Analysis of the Pigment Cell Specific Gene MART-1/Melan-A/mlana

Iraz Toprak Aydin

Pigmentation is a complex process that provides many diverse benefits for the organism. Pigment production takes place in the pigment cell lineage in dedicated organelles called melanosomes. Several proteins are specifically recruited to melanosomes for this purpose, and many of them are involved in melanin synthesis. MART-1 is one of the melanosomal proteins. It has been widely studied as an agent for immunotherapy against malignant melanoma but very little is known about the gene and its function. Here, I used different approaches in order to understand the biology of this pigment cell specific gene. First, I analyzed transcriptional regulation of the mouse MART-1 gene. I found that the shortest promoter region (0.6kb) alone is not sufficient to drive the expected MART-1 expression in vivo, even though it was able to express a luciferase reporter in vitro, in transfected cells. The largest promoter region (6.0kb) was able to produce expression of a LacZ reporter gene in the RPE layer starting from the early stages of embryogenesis. However it failed to drive melanoblast-specific expression in embryos and, only after birth, reporter gene expression in melanocytes became visible, indicating that distal regulatory elements might be involved in melanocyte/melanoblastspecific MART-1 expression. In order to visualize the spatial and temporal expression pattern of MART-1, I developed a MART-1::LacZ BAC reporter mouse and showed that in mouse, MART-1 expression starts at around E11.5-12.5, and its expression is exclusive to melanocytes and RPE. To understand the function of MART-1, I developed a knockout mouse model. I have shown for the first time that MART-1 is directly involved in pigmentation and that its loss leads to coat color dilution. It has been reported that MART-1 might be involved in trafficking and processing of the melanosomal proteins Pmel17 and OA1. I analyzed the distribution of Pmel17 and OA1, as well as other melanosomal proteins in melanocyte lines derived from MART-1 knockout mice, however no mislocalizations were observed. Besides, in primary knockout melanocytes, the processing of Pmel17 was not affected. Nevertheless, electron microscopy studies revealed that MART-1 knockout mice had severe morphological defects in melanosomes of the hair follicle melanocytes. This indicated that the coat color dilution observed in MART-1 knockout mice might be due to the structural defects in the melanosomes, suggesting that MART-1 is required for melanosome biogenesis in the hair follicle.

EPFL2010

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice

Friedrich Beermann

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.

2004

Murine Neonatal Melanocytes Exhibit a Heightened Proliferative Response to Ultraviolet Radiation and Migrate to the Epidermal Basal Layer

Friedrich Beermann

Melanocytes respond to UVR not only by producing melanin, but also by proliferating. This is essentially a protective response. We have studied the melanocyte proliferative response after a single UVR exposure to neonatal mice. At 3 days post-UVR in wild-type neonates we observed a marked melanocyte activation not seen in adults. Melanocytes migrated to the epidermal basal layer, their numbers peaking at 3-5 days after UVR then diminishing. They appeared to emanate from the hair follicle, migrating to the epidermis via the outer root sheath. In melanoma-prone mice with melanocyte-specific overexpression of Hras(G12V), basal layer melanocytes were increased in size and dendricity compared to UVR-treated wild-type mice. Melanocytes in mice carrying a pRb pathway cell-cycle defect (oncogenic Cdk4(R24C)) did not show an enhanced response to UVR such as those carrying Hras(G12V). The exquisite sensitivity to UVR-induced proliferation and migration that characterizes neonatal mouse melanocytes may partly explain the utility of this form of exposure for inducing melanoma in mice that carry oncogenic mutations

2009

Analysis of the Pigment Cell Specific Gene MART-1/Melan-A/mlana

Iraz Toprak Aydin

EPFL2010