Investigating Functional Ependymal Cell Heterogeneity in the Ventricular System

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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.