Unraveling Cochlear Otic Mesenchyme Cells: The Role of Pou3f4 in Cochlear Development

项目摘要 听力障碍是一个日益严重的医疗保健问题，它可能对个人的听力质量产生很大影响。 生活耳蜗是由一个大的异质细胞群，所有的工作在和谐转换机械 将刺激转化为电信号，从而产生声音的感知。一种这样的细胞类型是耳间充质 细胞，耳蜗内数量最多的细胞类型，对于正常听力的成熟至关重要 在人类和小鼠中。事实上，Pou 3f 4（一种间充质特异性转录因子）的突变导致了一种新的细胞因子的产生。 发育中耳蜗的各种缺陷，包括耳蜗内电位的完全丧失， 耳蜗管，螺旋神经节神经元（SGN）的存活和成束减少。我们最近 使用scRNA-seq发现，耳间充质细胞不是同质群体，但可以是 分为4个基因独特的亚群。我们还显示，使用免疫组织化学， 对于CAR 3和TGFBI的抗体，四个间充质亚群中的两个在空间上不同， 耳蜗，显示Car 3阳性的耳间充质细胞位于外侧壁内，围绕耳蜗。 血管纹和Tgfbi阳性的耳间充质细胞组成螺旋状利姆布斯，这是SGN所必需的 寻路因此，我们推测耳蜗间充质细胞由几个亚群组成 它们是（a）空间上不同和（B）调节共有和独特的下游靶基因。为了测试这种 假设，我们设计了两个目标：（1）确定耳蜗间充质的空间分布 亚群在Pou 3f 4缺失的情况下发生变化，以及（2）定义区域基因调控网络 在耳间充质细胞中Pou 3f 4的下游。使用完善的体外小鼠模型和三个- 单细胞多组学方法，我们将阐明每个间充质细胞中的调控元件， 亚群，导致不仅发现耳间充质成熟所必需的基因， 关键耳蜗过程（如耳蜗内电位）发育所必需的基因。这项建议 是发展耳蜗成熟的更完整理解的垫脚石， 了解对正常听力至关重要的未充分研究的细胞类型的作用。

PROJECT SUMMARY Hearing impairment is a growing healthcare issue, which can have a large impact on an individual’s quality of life. The cochlea is made up of a large heterogenous group of cells, all working in harmony to convert mechanical stimuli into electrical signals resulting in the perception of sound. One such cell type is the otic mesenchyme cells, the most numerous cell type within the cochlea, which are essential for the maturation of normal hearing in both human and mouse. Indeed, mutations in Pou3f4, a mesenchymal specific transcription factor, causes a variety of defects in the developing cochlea, including complete loss of endocochlear potential, shortening of the cochlear duct, and decreased survival and fasciculation of spiral ganglion neurons (SGNs). We have recently discovered, using scRNA-seq, that otic mesenchyme cells are not a homogenous population, but can be separated into 4 genetically unique subpopulations. We have also shown, using immunohistochemistry with antibodies for CAR3 and TGFBI, that two of the four mesenchyme subpopulations are spatially distinct within the cochlea, revealing Car3-postive otic mesenchyme cells are located within the lateral wall, surrounding the stria vascularis, and Tgfbi-positive otic mesenchyme cells make up the spiral limbus, which is essential for SGN pathfinding. Therefore, we hypothesize that the cochlear mesenchyme cells consist of several subpopulations that are (a) spatially distinct and (b) regulate shared and unique downstream target genes. In order to test this hypothesis, we have designed two aims: (1) Determine how the spatial distribution of the cochlear mesenchyme subpopulations are changed in the absence of Pou3f4 and (2) Define the regional gene regulatory networks downstream of Pou3f4 in otic mesenchyme cells. Using a well-established in vitro mouse model and a three- pronged single-cell multi-omics approach, we will elucidate regulatory elements in each mesenchymal subpopulation, leading to the discovery of not only genes necessary for otic mesenchyme maturation, but novel genes essential for the development of key cochlear processes such as endocochlear potential. This proposal is a steppingstone in developing a more complete understanding of cochlear maturation, and specifically understanding the role of an understudied cell type crucial for normal hearing.