Investigating Functional Ependymal Cell Heterogeneity in the Ventricular System

项目摘要/摘要： 在脊椎动物的大脑中，神经胶质细胞的总数超过了神经元，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)及其许多活动的纤毛就是其中之一 神秘的神经胶质细胞群。即使与其他类型的神经胶质细胞相比，人们对它们的了解也相对较少。研究项目： Alvarez-Buylla实验室：小鼠已经证明室管膜细胞对正常的大脑功能是必不可少的 另一些研究表明，室管膜平面细胞极性的缺陷或纤毛跳动干扰了脑脊液的流动。这些 动物会患上脑积水，由于脑积水增加而导致广泛的脑损伤。 颅内压。然而，室管膜细胞作为脑脊液管道的观点已经被证明是过度的。 简单化了。众所周知，出生后啮齿动物大脑中最大的神经源性生态位--脑室-- 室下区(V-SVZ)嵌于侧脑室侧壁。室管膜细胞形成一种 接触脑脊液的成体神经干细胞(ANSCs)周围有风车状结构。我们实验室以前的工作 已表明室管膜细胞通过Noggin信号调节神经干细胞的神经发生。最近，该实验室已经 描述了一个存在于所有脑室的在形态上不同的室管膜细胞，它只有两个活动纤毛。 结构不典型的纤毛基底体。令人惊讶的是，在第四脑室中，双纤毛细胞延伸出一条长的 从脑室表面到中缝背核(DRN)的基底突起是 大脑中的血清素。室管膜细胞镶嵌在V-SVZ中，调节其神经发生活动。然而，a 知识中的关键差距是我们对室管膜细胞调节细胞功能的理解 他们直接接触的脑室周围脑区，如DRN。取得进展的关键障碍 了解室管膜细胞的异质性是缺乏分子标记和工具来独立 操纵室管膜细胞亚群。为了克服这些障碍，我创造了一个单细胞 来自V-SVZ的测序数据集，并分析了来自公共的室管膜细胞和DRN神经元 可用的单细胞测序数据集。根据初步数据，我假设室管膜细胞是一种 转录异质性群体，在V-SVZ和第四脑室有不同的功能作用。 在这份提案中，我提出了两个相互垂直的目标，横跨K99和R00两个阶段的奖项。在第一个 目的应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里。R00阶段主要是在目标2中完成的，在那里我将在我现有的初步单曲的基础上进行构建 细胞RNA测序分析对室管膜功能异质性的机械性洞察 第四脑室的细胞。总而言之，这些数据将为室管膜如何 整个脑室系统中的细胞对局部大脑功能有贡献。

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.