Transcription profiling of mouse hairless mutant Hos: HR-1 to investigate mechanism alopecia

The Hos: HR-1 mouse strain (HR-1) is an autosomal resessive mutant strain which is widely used in pharmacological and dermatological studies in Japan. Although HR-1 mice have been believed to be Hr gene mutants based on their distinctive alopecia phenotype, there are no reports on either the genetic nature of the Hr gene or mechanism of alopecia. The present study clarified that the HR-1 mice carry a C to T transition in the Hr gene, which causes a proline to serin amino acid change in the functional domain of translated HR protein. It is well known that HR plays an important role in hair follicle regression, and loss of HR function triggers distinctive alopecia with the presence of premature apoptosis in the regressing hair follicle. However, the detailed function of HR protein is still uncertain. To clarify the mechanism by which HR dysfunction causes hair loss, here we performed microarray analysis on the mRNA samples obtained from the regressing hair follicles of the homozygous (HrHos / HrHos) and heterozygous (+/HrHos, control) Hr mutant mice using laser capture microdissection. Our results showed increased expression of apoptosis-related and cell-cycle-repressive genes and decreased expression of cell-cycle-promoting genes, suggesting inhibition of the cell cycle at G0/G1. These findings strongly suggest that the loss of HR function leads to high expression of cell cycle inhibitors, resulting in the hair loss with premature apoptosis in the hair matrix. Experiment Overall Design: Homozygous (HrHos / HrHos) and heterozygous (+/HrHos) Hr mutant mice of the same N1 littermates obtained from a male Hos: HR-1 mouse and a female heterozygous F1 offspring (+/HrHos) between an HR-1 mouse and an ICR mouse were used at day 14 postpartum. +/HrHos mice were used as controls because the Hr gene mutantion is autosomal recessive and the morphological appearance of +/HrHos is as same as that of wild-type mice. Four samples (two from +/HrHos and two from HrHos / HrHos) were analyzed. All RNA samples were extracted from hair follicles of dorsal skin using Laser capture microdissection.Hos: HR-1小鼠品系（HR-1）是一种常染色体隐性（autosomal recessive）突变品系，在日本广泛应用于药理学与皮肤病学研究。尽管基于HR-1小鼠独特的脱毛表型，学界普遍认为其属于Hr基因突变小鼠，但目前尚无关于Hr基因的遗传本质或脱毛机制的相关报道。本研究明确了HR-1小鼠的Hr基因存在C到T的碱基转换，该突变会导致翻译产生的HR蛋白功能结构域中脯氨酸突变为丝氨酸。已知HR在毛囊退行过程中发挥重要作用，HR功能缺失会引发特征性脱毛，并伴随退行期毛囊中的过早细胞凋亡。但目前HR蛋白的具体功能仍不明确。为阐明HR功能异常引发脱发的具体机制，本研究采用激光捕获显微切割（Laser Capture Microdissection）技术，对纯合型（HrHos / HrHos）与杂合型（+/HrHos，作为对照）Hr突变小鼠的退行期毛囊提取的mRNA样本进行了基因芯片（microarray）分析。研究结果显示，与凋亡相关及细胞周期抑制相关的基因表达上调，而细胞周期促进相关基因表达下调，提示细胞周期在G0/G1期受到阻滞。上述结果强烈表明，HR功能缺失会导致细胞周期抑制剂的高表达，进而引发毛基质中过早细胞凋亡并最终导致脱发。 实验总体设计：本研究使用的纯合型（HrHos / HrHos）与杂合型（+/HrHos）Hr突变小鼠均为同批N1代同窝仔鼠，其亲本为雄性Hos: HR-1小鼠，以及由HR-1小鼠与ICR小鼠杂交得到的雌性杂合型F1后代（+/HrHos），实验取材时间为产后第14天。由于Hr基因为常染色体隐性遗传，且杂合型（+/HrHos）小鼠的形态表型与野生型小鼠一致，因此以+/HrHos小鼠作为对照。本研究共分析4份样本（其中2份来自+/HrHos小鼠，2份来自HrHos / HrHos小鼠）。所有RNA样本均通过激光捕获显微切割技术，从背部皮肤的毛囊中提取得到。