Data_Sheet_1_Vestibular and Auditory Hair Cell Regeneration Following Targeted Ablation of Hair Cells With Diphtheria Toxin in Zebrafish.PDF

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

数百万美国人因内耳毛细胞（hair cells）丢失而罹患听力或平衡障碍。毛细胞是所有脊椎动物听觉与前庭器官，以及水生脊椎动物侧线系统（lateral line systems）中发挥功能的机械感觉受体。在斑马鱼（zebrafish）与其他非哺乳类脊椎动物体内，毛细胞会在稳态维持过程中发生更新，并在受到声学或化学暴露导致损伤或破坏后完全再生。然而在哺乳动物体内，毛细胞受损或丢失会引发永久性听力或平衡功能损伤。本研究旨在开发一种用于研究成年水生脊椎动物毛细胞损伤与再生的优化方法，为此构建了可在体内实现靶向诱导性毛细胞消融的转基因斑马鱼。该模型以myo6b启动子（myo6b promoter）调控人类白喉毒素受体（human diphtheria toxin receptor, hDTR）基因的表达，使hDTR仅在毛细胞中产生。通过向成年斑马鱼腹腔注射白喉毒素（diphtheria toxin, DT），或向幼体养殖水体中添加溶解有DT的溶液，即可实现毛细胞消融。在受精后5天（days post fertilization, dpf）的斑马鱼侧线中，DT处理引发的毛细胞消融可在2天内以剂量依赖的方式完成。类似地，针对成年椭圆囊（utricles）与球囊（saccules），单次腹腔注射0.05 ng DT可在注射后5天内导致椭圆囊与球囊内的毛细胞完全缺失。研究人员观察到侧线系统与内耳组织的毛细胞实现了完全再生。本研究介绍了一种可在成年斑马鱼内耳感觉上皮（椭圆囊与球囊）中实现高效条件性毛细胞消融的新方法，并证实经该处理后，斑马鱼毛细胞可在体内完成再生。