Investigating Functional Ependymal Cell Heterogeneity in the Ventricular System

项目概要/摘要： 神经胶质细胞在脊椎动物大脑中的总数超过神经元，但对其机制的理解 缺乏分子亚型和功能。室管膜细胞，排列在大脑中的纤毛上皮细胞 脑室和产生层流的脑脊液（CSF）与他们的许多运动纤毛，是这样一个 神经胶质的神秘群体。即使与其他神经胶质细胞类型相比，对它们的了解也相对较少。研究 小鼠已经证明室管膜细胞对正常的脑功能是必不可少的：阿尔瓦雷斯-布伊拉实验室 其他人已经表明室管膜平面细胞极性或纤毛搏动的缺陷破坏CSF流动。这些 动物发展为脑积水，由于增加的脑内毒素， 颅内压然而，室管膜细胞作为脑脊液管道的观点已被证明是过度的。 太简单了众所周知，出生后啮齿动物大脑中最大的神经原性龛，脑室- 脑室下区（V-SVZ）嵌入侧脑室的侧壁中。室管膜细胞形成 在接触CSF的成体神经干细胞（aNSC）周围的风车状结构。我们实验室以前的工作 已经显示室管膜细胞通过Noggin信号传导调节aNSC神经发生。最近，该实验室 描述了存在于所有脑室中的一种形态学上独特的室管膜细胞，它只有两条运动纤毛 和结构上非典型的纤毛基体。引人注目的是，在第四脑室，双纤毛细胞延伸出一个长的 中缝背核（Dorsal Raphe Nucleus，DRN）是脑组织的主要来源。 大脑中的血清素室管膜细胞包埋在V-SVZ调节其神经活性。但 关键的知识差距是我们对室管膜细胞调节其他细胞功能的理解。 它们直接接触的脑室周围脑区，如DRN。一个关键的障碍， 理解室管膜细胞异质性是缺乏分子标记和工具， 操纵室管膜细胞的亚群。为了克服这些障碍，我创造了一个单细胞 测序数据集从V-SVZ，并分析了室管膜细胞和DRN神经元从公开的 单细胞测序数据集。基于初步的数据，我假设室管膜细胞是一种 在V-SVZ和第四脑室中具有不同功能作用的转录异质群体。 在这个建议中，我提出了两个正交的目标，跨越K99和R 00阶段的奖励。上 目的利用单细胞测序技术鉴定室管膜细胞和成体神经干细胞的异质性 在V-SVZ R 00阶段主要是在目标2中完成的，在那里我建立在我现有的初步单 细胞RNA测序分析，以获得对室管膜功能异质性的机制洞察 第四脑室的细胞总之，这些数据将提供一个基本的了解室管膜如何 整个脑室系统的细胞对局部脑功能有贡献。

Project Summary/Abstract: Glial cells collectively outnumber neurons in the vertebrate brain, but mechanistic understanding of their molecular subtypes and functions is lacking. Ependymal cells, ciliated epithelial cells that line the brain ventricles and produce laminar flow of cerebral spinal fluid (CSF) with their many motile cilia, are one such enigmatic group of glia. Relatively little is known about them, even compared to other glial cell types. Studies in mice have demonstrated that ependymal cells are essential for normal brain function: the Alvarez-Buylla lab and others have shown that defects in ependymal planar cell polarity or ciliary beating disrupt CSF flow. These animals develop hydrocephalus, causing widespread damage throughout the brain due to increased intracranial pressure. The view of ependymal cells as CSF conduits, however, has proven to be overly simplistic. It is known that the largest neurogenic niche in the postnatal rodent brain, the ventricular- subventricular zone (V-SVZ) is embedded in the lateral walls of the lateral ventricles. Ependymal cells form a pinwheel-like structure around the CSF-contacting adult neural stem cells (aNSCs). Previous work from our lab has shown that ependymal cells regulate aNSC neurogenesis via Noggin signaling. More recently, the lab has described a morphologically distinct ependymal cell present in all brain ventricles that has only two motile cilia and structurally atypical ciliary basal bodies. Strikingly, in the fourth ventricle the bi-ciliated cells extend a long basal process from the ventricular surface into the Dorsal Raphe Nucleus (DRN), the primary source of serotonin in the brain. Ependymal cells embedded in the V-SVZ regulate its neurogenic activity. However, a critical gap in knowledge is our understanding of ependymal cell capacity to regulate the function of other periventricular brain areas they directly contact, such as the DRN. A critical barrier to progress in understanding ependymal cell heterogeneity is the lack of molecular markers and tools to independently manipulate subpopulations of ependymal cells. To overcome these barriers, I have generated a single-cell sequencing dataset from the V-SVZ and have analyzed ependymal cells and DRN neurons from a publicly available single-cell sequencing dataset. Based on preliminary data, I hypothesize that ependymal cells are a transcriptionally heterogeneous population, that have distinct functional roles in the V-SVZ and fourth ventricle. In this proposal, I put forward two orthogonal Aims that span the K99 and R00 phases of the award. In the first Aim I use single cell sequencing to identify heterogeneity among ependymal cells and adult neural stem cells in the V-SVZ. The R00 phase is mainly accomplished in Aim 2, where I build on my existing preliminary single cell RNA-sequencing analyses to gain mechanistic insight into functional heterogeneity among ependymal cells in the fourth ventricle. Together, these data will provide a foundational understanding of how ependymal cells throughout the ventricular system contribute to local brain function.