Characterization of the transcriptomes of Atoh1-induced hair cells in the mouse cochlea. Characterization of the transcriptomes of Atoh1-induced hair cells in the mouse cochlea

Mammalian cochlea hair cells can be regenerated from the adjacent supporting cells in many ways, however, the newly hair cells are immature and without hearing function almost. The expression of Atoh1 is an important maker of the appearance of newly hair cells, many approaches of inner ear hair cells’ regeneration are concerned with the transcription factor Atoh1. However, most new HCs are immature HCs, and they do not have the function of mature HCs and eventually die a few weeks after regeneration. Thus promoting the maturation and survival of new HCs is the primary focus of HC regeneration field. We used RNA-Seq analysis to compare the differences between the transcriptomes of Atoh1 overexpression-induced new HCs and the original HCs, and to define the factors that might help to promote the maturation and survival of new HCs. Overall design: We used transgenic mice in the C57BL/6J background to perform this assay. Sox2-CreER mice (JAX number 008875) and Rosa26-tdTomato mice (JAX number 007914) were ordered from the Jackson Laboratory. Atoh1-eGFP (enhanced green fluorescent protein) mice were provided by Jane Johnson (University of Texas Southwestern Medical Center, Dallas, TX, USA), and CAG-loxP-stop-loxP-Atoh1-HA+ mice (Atoh1-HA+ mice) were the kind gift of St. Jude Children's Research Hospital. Postnatal day (P) 0 was defined as the day of birth. Both male and female mice were used for all experiments. Tamoxifen (Sigma-Aldrich) diluted in corn oil was injected intraperitoneally at the late stage of P3 at 0.20 mg/g bodyweight, and the control group was injected only with corn oil. Sox2-CreER+/Atoh1-HA+/Atoh1-eGFP+/tdTomato+ fourth positive mice were sacrificed at P7, and the cochlear epithelium was dissected and trypsinized with pre-warmed 0.25% trypsin/EDTA (Invitrogen) at 37°C for 5 min. Soybean trypsin inhibitor (Worthington Biochem) was added to terminate the reaction followed by mechanical trituration with blunt tips and pipetting up and down ~100 times. Suspended cells were percolated through a 40 µm cell strainer (BD Biosciences) before FACS. The original HCs were labeled with enhanced green fluorescent protein (eGFP+ cells), new HCs were co-labeled with enhanced green fluorescent protein and tdTomato red fluorescent protein (eGFP+/tdTomato+ cells), and SCs were labeled with tdTomato red fluorescent protein (tdTomato+ cells). The original HCs, new HCs, and SCs were sorted on a BD FACS Aria III (BD Biosciences) using the tdTomato and GFP channels. The samples are prepared for the following experiments including PCR and RNA sequence analysis.

哺乳动物耳蜗毛细胞可通过多种途径由邻近的支持细胞再生，但新生毛细胞多处于未成熟状态，几乎不具备听觉功能。Atoh1（Atonal bHLH转录因子1）是新生毛细胞出现的重要标志物，内耳毛细胞再生的诸多策略均与该转录因子相关。然而，绝大多数新生毛细胞仍为未成熟表型，无法发挥成熟毛细胞的功能，且在再生数周后最终凋亡。因此，促进新生毛细胞的成熟与存活，是当前毛细胞再生研究领域的核心焦点。

我们通过RNA测序（RNA-Seq）分析，比较了Atoh1过表达诱导的新生毛细胞与原生毛细胞的转录组差异，以筛选可助力新生毛细胞成熟与存活的关键因子。

总体实验设计：本研究使用C57BL/6J背景的转基因小鼠开展实验。Sox2-CreER小鼠（JAX品系编号：008875）与Rosa26-tdTomato小鼠（JAX品系编号：007914）购自杰克逊实验室（Jackson Laboratory）；Atoh1-增强型绿色荧光蛋白（Atoh1-eGFP）小鼠由美国德克萨斯大学西南医学中心Jane Johnson教授馈赠；CAG-loxP-stop-loxP-Atoh1-HA+小鼠（简称Atoh1-HA+小鼠）由圣犹大儿童研究医院惠赠。本研究将出生当日记为出生后第0天（P0），所有实验均使用雌雄各半的小鼠。

于出生后第3天（P3）晚期，向小鼠腹腔注射溶于玉米油的他莫昔芬（Sigma-Aldrich），注射剂量为0.20 mg/g体重；对照组仅注射玉米油。将同时携带Sox2-CreER+、Atoh1-HA+、Atoh1-eGFP+与tdTomato+的四阳性小鼠于出生后第7天（P7）时处死，分离耳蜗上皮，用预热至37℃的0.25%胰蛋白酶-乙二胺四乙酸（Invitrogen）消化5分钟，随后加入大豆胰蛋白酶抑制剂（Worthington Biochem）终止消化反应，再使用钝头吸管进行约100次上下吹打以完成机械解离。将细胞悬液通过40 μm细胞筛（BD Biosciences）过滤后，进行荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）。原生毛细胞通过增强型绿色荧光蛋白标记（eGFP+细胞），新生毛细胞同时被增强型绿色荧光蛋白与tdTomato红色荧光蛋白标记（eGFP+/tdTomato+细胞），支持细胞则仅被tdTomato红色荧光蛋白标记（tdTomato+细胞）。通过BD FACS Aria III流式细胞仪（BD Biosciences）的tdTomato与GFP通道，分选出原生毛细胞、新生毛细胞与支持细胞，制备样本用于后续聚合酶链式反应（PCR）与RNA测序分析。