Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons.

哺乳动物内耳神经感觉细胞的命运由三种密切相关的转录因子（transcription factor）决定：调控毛细胞的Atoh1，以及调控神经元的Neurog1和Neurod1。我们此前已证实，通过消除神经元中Neurod1介导的Atoh1表达抑制作用，可将神经元细胞命运向毛细胞命运重编程。为验证毛细胞命运决定过程中是否存在类似的细胞可塑性，我们构建了敲入（knockin, KI）小鼠品系（Atoh1KINeurog1），该品系中Atoh1被Neurog1替换。在Atoh1启动子的调控下表达Neurog1，会改变杂合子（Atoh1+/KINeurog1）与纯合子（Atoh1KINeurog1/KINeurog1）KI小鼠的毛细胞前体细胞的基因表达模式、分化进程与存活状态。纯合子KI小鼠可形成表达Neurog1、Neurod1、多种感前体细胞基因及神经营养因子的耳蜗螺旋器（organ of Corti）前体细胞斑块；此外，这些细胞斑块可接收传入与传出神经突起。该斑块中的部分细胞可形成多根微绒毛，但无法形成静纤毛（stereocilia）。值得注意的是，表达Neurog1的突变体与Atoh1基因敲除突变体存在差异：前者可间歇性形成类耳蜗螺旋器斑块，而后者在缺失Atoh1时会形成完全的“扁平上皮”。在杂合子KI小鼠中，Atoh1与Neurog1的共表达不仅会导致部分毛细胞与支持细胞的命运及模式发生改变，还会在发育后期引发毛细胞极性异常。这一现象与Atoh1单倍体剂量不足（haploinsufficiency）模型（Pax2cre; Atoh1f/+）存在差异，表明Neurog1在发育中的毛细胞中发挥了特定调控作用。我们的研究数据表明，Atoh1KINeurog1可在一定程度上为耳蜗螺旋器细胞的存活提供功能支持。与此前证实的神经元可向毛细胞分化的命运可塑性不同，毛细胞前体细胞可在Neurog1的作用下维持有限时长，但无法向神经元转分化（transdifferentiate）。