小鼠和斑马鱼内耳支持细胞转分化的调控机制研究

Mammalian hair cells are neuroepithelial cells characterized by apical bundles composed of numerous actin-filled stereocilia. These mechanosensory cells are lost with age and are highly susceptible to damage from noise, and ototoxic drugs. This age- and trauma-induced susceptibility is a profound health problem because a significant proportion of the human population suffers from deafness or balance disorders resulting directly from hair cell loss. For this obvious reason, there have been enormous interests in hair cell regeneration since late 80’s after Rubel, Corwin, Cotanche, and colleagues discovered that bird hair cells were able to regenerate. We now know that the auditory and vestibular organs of many non-mammalian vertebrates such as fish, amphibians, and birds retain the capacity to regenerate hair cells through proliferation and transdifferentiation of supporting cells after acoustic and aminoglycoside-induced damage to existing hair cells. This ability, however, is lost in mammalian hair cells and supporting cells since they become postmitotic during embryonic days and remain without proliferation and regeneration after birth. The molecular mechanisms of why non-mammalian supporting cells are able to proliferate and transdifferentiate while mammalian supporting cells are completely unclear. Understanding the gene expression profiles (i.e., transcriptomes) of non-mammalian and mammalian supporting cells hold the key to understand the molecular mechanisms of hair cell regeneration. The transcriptome reflects the genes that are being actively expressed in a cell and is critical for understanding the molecular mechanisms of the biological properties of the cell. We propose to analyze the transcriptomes of inner ear supporting cells in adult mice as well as in transgenic zebrafish supporting cells with GFP expression. There are several types of supporting cells in the mammalian cochlea, but pillar and Deiters’cells are morphologically distinct and retain some capacity to transdifferentiate to hair cell-like cells when Atoh1 is overexpressed. One thousand pillar cells, 1,000 Deiters’ cells, and 1000 zebrafish GFP-positive supporting cells will be individually collected from adult mice and zebrafish, using the suction pipette technique we pioneered. We will then use RNA-seq techniques to determine what genes are expressed in each cell type and what genes are uniquely or differentially expressed in mouse and zebrafish supporting cells. In Aim 1, we will determine the signal pathways of those differentially or uniquely expressed genes and the transcription factors between zebrafish and mouse inner ear supporting cells, using RNA-seq and bioinformatics. In Aim 2, we will use Crispr/Cas9 gene-editing technique to generate knockout mice and/or to use over-expression model in vitro (adenovirus carrying a target gene to transfect cultured organ of Corti) to determine whether altering the expression of this gene identified in Aim 1 will lead to conversion of supporting cells to new hair cells in adult mice. Our work will lay the groundwork for identification and characterization of genes that are critical for inner ear supporting cell proliferation and transdifferentiation, a crucial step for identifying target genes and pathways for hair cell regeneration using the gene therapy.

哺乳动物内耳毛细胞损失是造成感音性耳聋最主要的原因。非哺乳动物内耳的支持细胞在毛细胞损伤后，可转分化为具有功能的毛细胞，而哺乳动物内耳支持细胞则丧失了这种能力。我们推测这是由于哺乳动物支持细胞中某些调控基因（转录因子）限制了细胞的转分化。研究非哺乳和哺乳动物支持细胞的转录组是找到这些调控基因的关键。本研究拟用我们首创的单细胞收集技术，通过RNA-seq比较小鼠和转基因斑马鱼内耳支持细胞的转录组，来确定调控支持细胞转分化的基因和信号通路。明确候选基因后，我们将利用Crispr/Cas9条件性敲除小鼠模型和用腺病毒转染过表达基因离体模型，通过形态学、细胞电生理和分子生物学方法，来证实改变该基因的表达是否可以促使支持细胞转分化为毛细胞。此项研究将有望阐明成年哺乳动物支持细胞转分化为毛细胞的调控机制，并为最终使用基因疗法治疗感音性耳聋奠定实验基础。

哺乳动物内耳毛细胞损失是造成感音性耳聋最主要的原因。非哺乳动物内耳的支持细胞在毛细胞损伤后，可转分化为具有功能的毛细胞，而哺乳动物内耳支持细胞则丧失了这种能力。我们推测这是由于哺乳动物支持细胞中某些调控基因（转录因子）限制了细胞的转分化。研究非哺乳和哺乳动物支持细胞的转录组是找到这些调控基因的关键。本研究采用我们首创的单细胞收集技术，通过RNA-seq比较小鼠和转基因斑马鱼内耳支持细胞的转录组，来确定调控支持细胞转分化的基因和信号通路。明确候选基因后，我们将利用Crispr/Cas9条件性敲除小鼠模型和用腺病毒转染过表达基因离体模型，通过形态学、细胞电生理和分子生物学方法，来证实改变该基因的表达是否可以促使支持细胞转分化为毛细胞。此项研究将有望阐明成年哺乳动物支持细胞转分化为毛细胞的调控机制，并为最终使用基因疗法治疗感音性耳聋奠定实验基础。.我们完成了小鼠及斑马鱼内耳毛细胞及支持细胞转录组的获取、分析和比较。通过比较找到了共同表达和特异性表达的基因，并用原位杂交、抗体染色和PCR的方法验证了不同的基因在这些细胞中的表达。 确定了在斑马鱼和小鼠毛细胞中特异性表达的基因（如附件图1示），并用电生理的方法确定斑马鱼毛细胞上表达的动力蛋白（prestin）并不能象小鼠外毛细胞上表达的prestin一样产生电致运动 (motility)..我们也比较了斑马鱼支持细胞和小鼠支持细胞基因表达的异同，发现斑马鱼支持细胞有约1300基因是小鼠支持细胞上所没有表达的。这些基因包括许多调控基因表达的转录因子和控制细胞周期的基因。我们发现一个在小鼠及斑马鱼毛细胞中特异性表达的基因 Slc7a14，因此建立了Slc7a14敲除的小鼠，进一步的研究该基因的功能。我们的研究表明，该基因的变异可导致视网膜黄斑和耳聋，所以这个基因的变异可以导致综合症性耳聋。相关论文已经在修稿中。