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

项目总结 听力障碍是一个日益严重的医疗问题，它可能会对个人的听力质量产生很大影响 生活。耳蜗是由一大群异质细胞组成的，所有细胞都协调地工作以转换机械 刺激转化为电信号，导致对声音的感知。耳间充质细胞就是这样一种细胞类型。 细胞，耳蜗内数量最多的细胞类型，对正常听力的成熟是必不可少的 在人类和老鼠身上都有。事实上，间充质特异性转录因子Pou3f4的突变会导致 发育中的各种耳蜗缺陷，包括耳蜗内电位完全丧失，耳蜗管长度缩短 耳蜗管损伤，螺旋神经节神经元(SGN)存活和丛生减少。我们最近做了 使用scrna-seq发现，耳廓间充质细胞不是同质的群体，但可以是 分成4个遗传独特的亚群。我们还展示了，使用免疫组织化学 CAR3和TGFBI的抗体，四个间充质亚群中的两个在空间上是不同的 耳蜗，显示Car3阳性的耳间充质细胞位于侧壁内，包围着 血管纹和Tgfbi阳性的耳间充质细胞组成螺旋状缘缘，这是SGN所必需的。 寻路。因此，我们假设耳蜗间充质细胞由几个亚群组成。 它们是(A)空间上不同的和(B)调节共享的和独特的下游靶基因。为了测试这一点 假设，我们设计了两个目标：(1)确定耳蜗间充质的空间分布 在没有Pou3f4和(2)定义区域基因调控网络的情况下，亚群发生变化 Pou3f4下游的外耳间充质细胞。使用一个成熟的体外小鼠模型和一个三个- 采用单细胞多组学方法，我们将阐明每个间充质细胞中的调控元件 亚群，导致不仅发现了耳间充质成熟所必需的基因，而且发现了新的 对关键的耳蜗突发育至关重要的基因，如耳蜗内电位。这项建议 是发展对耳蜗成熟度的更全面的理解的一个踏脚石，特别是 了解未被充分研究的细胞类型对正常听力至关重要的作用。

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.