Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

Abstract

Oca2(p-cas) (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2(p-cas) revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.

Fig. 1.

External appearances of novel and ordinary Oca2^p-cas/Oca2^p-cas mutant mice on the non-agouti background. (A) Young novel mutant mouse at one month after birth before becoming darker in color. (B) Young ordinary mutant mouse at one month of age. (C) Adult novel mouse at 5 months of age (left side), which grew from the young mouse shown in (A). The eyes and coat hair became darker than those of the unchanged ordinary littermate (right side).

Fig. 2.

Cumulative frequency distribution of novel mutant mice that displayed darkening of the eyes. A total of 70 mice was observed.

Fig. 3.

Light micrographs of middle regions of dorsal awl hairs. (A) B6 non-agouti control mouse at about 4 months of age. (B) Ordinary Oca2^p-cas/Oca2^p-cas mutant mouse at about 4 months of age. (C) Novel Oca2^p-cas/Oca2^p-cas mutant mouse at 16 days of age. (D) The same novel mouse at about 4 months of age. Melanin pigmentation of the awl hair was obviously increased with aging. Scale bar=10 µm.

Fig. 4.

Hematoxylin-eosin-strained (A-C) and Fontana-Masson-stained light micrographs (D-F) of eyes from adult mice. (A, D) B6 control. (B, E) Ordinary mutant. (C, F) Novel mutant. Arrowheads show retinal pigment epithelium. Ch: choroid; Scale bar=50 µm.

Fig. 5.

Transmission electron micrographs of eyes from adult mice. (A) B6 control. (B) Ordinary mutant. (C) Novel mutant. Pigment accumulation (arrow) is visible in the choroid of the novel mutant mouse in (C). Sc: sclera; Ch: choroid; Rp: retinal pigment epithelium; Scale bar=10 µm.

Fig. 6.

Light micrographs of tail skin from adult mice stained with hematoxylin and eosin (A–C) and stained with Fontana and Masson (D–F). (A, D) B6 control. (B, E) Ordinary mutant. (C, F) Novel mutant. Insets indicate hair follicles that were used as positive controls for Fontana-Masson staining. Arrows indicate melanin pigmentation in the two types of mutant mice. De: dermis; Ep: epidermis; Sc: stratum corneum; Scale bar=20 µm.

Fig. 7.

Transmission electron micrographs of tail epidermis from adult mice. (A, D) B6 control. (B, E) Ordinary mutant. (C, F) Novel mutant. (D), (E) and (F) are magnified views of squared areas in (A), (B) and (C), respectively. Arrows show melanosomes. M: melanocyte; K: keratinocyte; F: fibroblast; Pc: prickle cell; Bm: basal membrane; Mi: mitochondria; Scale bar=1 µm.

Fig. 8.

Consequences of the Oca2^p-cas mutation. (A) Agarose gel electrophoretic patterns of RT-PCR products that were amplified eye cDNAs from B6 control mice and ordinary and novel mutant mice with primers mP-7 and mP-12 (Supplementary data 1) indicated by horizontal arrows. M: HyperLadder II (Bioline, London) molecular marker. (B) Schematic representation of the Oca2^p-cas deletion mutation shown on the cDNA sequence. Positions of the translation initiation codon (ATG) and stop codon (TAA) are shown by black arrowheads. White boxes denote each exon. Twelve gray boxes denote putative transmembrane domains. Direct sequencing of the RT-PCR products revealed a 281-bp deletion of exons 15 and 16. (C) Schematic representation of the Oca2^p-cas deletion mutation shown on the genomic DNA sequence. Black arrowheads represent 5′ and 3′ breakpoints of the deletion. Positions and directions of three primers, mP-17, mP-18 and mP-19 (Supplementary data 1), used for PCR amplification are indicated by horizontal arrows. Direct sequencing of the PCR products revealed a 4,140-bp deletion including exons 15 and 16.