Abstract
Microphthalmia-associated transcription factor (Mitf) is a key regulator for differentiation of melanoblasts, precursors to melanocytes. The mouse homozygous for the black-eyed white (Mitfmi-bw) allele is characterized by the white-coat color and deafness with black eyes due to the lack of melanocytes. The Mitfmi-bw allele carries LINE-1, a retrotransposable element, which results in the Mitf deficiency. Here, we have established the black spotting mouse that was spontaneously arisen from the homozygous Mitfmi-bw mouse lacking melanocytes. The black spotting mouse shows multiple black patches on the white coat, with age-related graying. Importantly, each black patch also contains hair follicles lacking melanocytes, whereas the white-coat area completely lacks melanocytes. RT-PCR analyses of the pigmented patches confirmed that the LINE-1 insertion is retained in the Mitf gene of the black spotting mouse, thereby excluding the possibility of the somatic reversion of the Mitfmi-bw allele. The immunohistochemical analysis revealed that the staining intensity for beta-catenin was noticeably lower in hair follicles lacking melanocytes of the homozygous Mitfmi-bw mouse and the black spotting mouse, compared to the control mouse. In contrast, the staining intensity for beta-catenin and cyclin D1 was higher in keratinocytes of the black spotting mouse, compared to keratinocytes of the control mouse and the Mitfmi-bw mouse. Moreover, the keratinocyte layer appears thicker in the Mitfmi-bw mouse, with the overexpression of Ki-67, a marker for cell proliferation. We also show that the presumptive black spots are formed by embryonic day 15.5. Thus, the black spotting mouse provides the unique model to explore the molecular basis for the survival and death of developing melanoblasts and melanocyte stem cells in the epidermis. These results indicate that follicular melanocytes are responsible for maintaining the epidermal homeostasis; namely, the present study has provided evidence for the link between melanocyte development and the epidermal microenvironment.
Highlights
Long interspersed element-1 (LINE-1 or L1) is a retrotransposable element that could cause various types of diseases [1]
The distribution of melanocytes in a pigmented hair follicle is similar to that seen in the wild-type C57BL/6J mouse [5], the number of pigmented hair follicles is limited in a given black patch of the black spotting mouse, as detailed below
The presence of non-pigmented hairs is responsible in part for the grayish tone of a black patch of the black spotting mouse (Fig 2), which may reflect the lower number of developing melanoblasts in the trunk region of the black-spotting mouse embryo at E12.5, compared to the control mouse embryo (Fig 11C)
Summary
Long interspersed element-1 (LINE-1 or L1) is a retrotransposable element that could cause various types of diseases [1]. LINE-1 is present in intron 3 of the microphthalmia-associated transcription factor (Mitf) gene of the black-eyed white (Mitfmi-bw) mouse [2]. The LINE-1 insertion results in the aberrant splicing of Mitf gene transcripts [2, 8], thereby decreasing the expression level of Mitf, including Mitf-M that is preferentially expressed in melanocyte-lineage cells [9, 10]. We have shown the Mitf-M expression in the projection neurons of the olfactory bulb [13]. These results suggest that Mitf-M expression may be regulated in neurons by a mechanism distinct from that in melanocyte-lineage cells. The influence of the LINE-1 insertion may be different between neurons and melanocyte-lineage cells
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