Casz1 is required for both inner hair cell fate stabilization and outer hair cell survival

Cochlear inner hair cells (IHCs) and outer hair cells (OHCs) require different transcription factors for their cell fate stabilization and survival, suggesting separate mechanisms are involved. Here, we found that the transcription factor Casz1 was crucial for early IHC fate consolidation and for OHC survival during mouse development. Loss of Casz1 resulted in transdifferentiation of IHCs into OHCs, without affecting OHC production. However, long-term OHC survival was compromised in Casz1 mutant mice. In addition, the transcription factor Gata3 was downregulated in Casz1-deleted IHCs and overexpressing Gata3 partially rescued IHC properties, OHC numbers, and hearing in Casz1-deleted mice. Thus, Casz1 plays critical roles in early IHC fate stabilization and OHC survival and could potentially provide a lead for therapies aimed at regenerating both IHCs and OHCs. Casz1 -/- IHCs were manually picked using the genetic mouse model: Atoh1Cre/+; Casz1flox/flox; Rosa26-CAG-Loxp-stop-Loxp-3xGFP(Ai47)/Rosa26-CAG-Frt-stop-Frt-tdTomato; Fgf8-Flpo/+. In this model, Casz1 -/- IHCs can be simultaneously labelled with GFP and Tdtomato. In total, 73 GFP+/Tdtomato+ Casz1-/- IHCs at P14 (P14_Casz1-/- IHCs in brief) were manually picked under the fluorescent microscope. The de-novo picked 73 Casz1-/- IHCs at P14 along with the 55 wild type IHCs at P14 in our previous study (GSE199369), were pooled together for further smart-seq based single-cell transcriptomic assay.耳蜗内毛细胞（inner hair cells, IHCs）与外毛细胞（outer hair cells, OHCs）的细胞命运稳定与存活依赖不同的转录因子，提示二者的调控机制存在差异。本研究发现，转录因子Casz1对小鼠发育过程中早期内毛细胞的命运巩固以及外毛细胞的存活至关重要。Casz1缺失可导致内毛细胞转分化为外毛细胞，且不影响外毛细胞的生成。但Casz1突变小鼠的外毛细胞长期存活能力受损。此外，在Casz1敲除的内毛细胞中，转录因子Gata3的表达水平下调；过表达Gata3可部分挽救Casz1敲除小鼠的内毛细胞特性、外毛细胞数量及听力功能。综上，Casz1在早期内毛细胞命运稳定与外毛细胞存活过程中发挥关键作用，可为同时实现内毛细胞与外毛细胞再生的治疗策略提供研究线索。 本研究通过基因工程小鼠模型Atoh1Cre/+; Casz1flox/flox; Rosa26-CAG-Loxp-stop-Loxp-3xGFP(Ai47)/Rosa26-CAG-Frt-stop-Frt-tdTomato; Fgf8-Flpo/+，手动分选获得Casz1敲除的内毛细胞。该模型中，Casz1-/-内毛细胞可同时被GFP与TdTomato标记。最终，我们在荧光显微镜下手动分选得到73株出生后第14天（P14）的GFP+/TdTomato+ Casz1-/-内毛细胞（简称P14_Casz1-/- IHCs）。将本次新分选得到的73株P14时期Casz1敲除内毛细胞，与本团队前期研究（GSE199369）中的55株P14时期野生型内毛细胞合并，通过基于smart-seq的单细胞转录组分析开展后续实验。