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. 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.