Table_2_Alteration of Musashi1 Intra-cellular Distribution During Regeneration Following Gentamicin-Induced Hair Cell Loss in the Guinea Pig Crista Ampullaris.xlsx

The mechanism underlying hair cell (HC) regeneration in the mammalian inner ear is still under debate. Understanding what molecules regulate the HC regeneration in mature mammals will be the key to the treatment of the inner ear disorder. Musashi1 (MSI1) is an RNA binding protein associated with asymmetric division and maintenance of stem cell function as a modulator of the Notch-1 signaling pathway. In this study, we investigated the cellular proliferative activity and changes in spatiotemporal pattern of MSI1 expression in the gentamicin (GM)-treated crista ampullaris (CA) in guinea pigs. Although the vestibular HCs in the CA almost disappeared at 14 days after injecting GM in the inner ear, the density of vestibular HCs spontaneously increased by up to 50% relative to controls at 56 days post-GM treatment (PT). The number of the type II HCs was significantly increased at 28 days PT relative to 14 days PT (p < 0.01) while that of type I HCs or supporting cells (SCs) did not change. The number of SCs did not change through the observational period. Administration of bromodeoxyuridine with the same GM treatment showed that the cell proliferation activity was high in SCs between 14 and 28 days PT. The changes in spatiotemporal patterns of MSI1 expression during spontaneous HC regeneration following GM treatment showed that MSI1-immunoreactivity was diffusely spread into the cytoplasm of the SCs during 7–21 days PT whereas the expression of MSI1 was confined to the nucleus of SCs in the other period. The MSI1/MYO7A double-positive cells were observed at 21 days PT. These results suggest that regeneration of vestibular HCs might originate in the asymmetric cell division and differentiation of SCs and that MSI1 might be involved in controlling the process of vestibular HC regeneration.

哺乳动物内耳毛细胞（hair cell, HC）的再生机制至今仍存在学术争议。阐明调控成熟哺乳动物毛细胞再生的分子机制，将是攻克内耳疾病治疗难题的核心关键。Musashi1（MSI1）是一类RNA结合蛋白，作为Notch-1信号通路的调控因子，参与细胞不对称分裂并维持干细胞功能。本研究以豚鼠为实验模型，探究了庆大霉素（gentamicin, GM）处理后其壶腹嵴（crista ampullaris, CA）内的细胞增殖活性，以及Musashi1表达的时空分布模式变化。在内耳注射庆大霉素14天后，壶腹嵴内的前庭毛细胞几乎完全消失；而在庆大霉素处理后56天（post-GM treatment, PT），前庭毛细胞密度较对照组自发回升至多50%。相较于庆大霉素处理后14天，处理后28天时II型毛细胞数量显著升高（p < 0.01），而I型毛细胞与支持细胞（supporting cells, SCs）的数量未发生明显改变。整个观察周期内，支持细胞的数量均无显著变化。联合给予溴脱氧尿苷与庆大霉素处理的实验结果显示，在庆大霉素处理后14至28天期间，支持细胞的增殖活性处于较高水平。庆大霉素处理后前庭毛细胞自发再生过程中，Musashi1表达的时空模式发生显著改变：在处理后7至21天，Musashi1免疫反应性弥散分布于支持细胞的细胞质中；而在其余时段，Musashi1的表达仅局限于支持细胞的细胞核内。在庆大霉素处理后21天时，可观测到Musashi1与肌球蛋白VIIA（MYO7A）双阳性细胞。上述结果表明，前庭毛细胞的再生可能起源于支持细胞的不对称分裂与分化，而Musashi1可能参与调控前庭毛细胞的再生进程。