Best Pediatric Basic Science Abstract: Single Cell Transcriptomic Atlas of CSF-interacting Cells in the Developmental Brain Reveals Active Crosstalk Among Distinct Cell Populations

Introduction: Cerebrospinal fluid (CSF) plays a critical role in the growth and functioning of the central nervous system (CNS). Traditionally, it's been assumed that CSF flows through the brain and its surrounding spaces as a uniform medium. Yet, it remains unclear if certain cells within the CNS have a more direct relationship with CSF, and what specific molecular and biophysical processes facilitate such interactions. Here, we characterize the brain-CSF interface across neurodevelopment using the first-ever single-cell transcriptomic atlas of CSF-interacting brain cells.

Methods: We infused CellTracker CMFDA fluorescent tracer into the mouse cisterna magna at postnatal days 8 (P8) and 21 (P21), then FACS-sorted the brain cells that interacted with and internalized the tracer. Through integrative single-cell RNA sequencing (ScRNA-seq) analysis and immunofluorescence imaging, we dissected the cellular and molecular mechanisms underlying CSF's influence on CNS development. We utilized CellChat, a ScRNA-seq tool, to infer intercellular ligand-receptor communication networks and characterize the crosstalk between cell clusters.

Results: CNS cell populations at P8 and P21 demonstrated significantly different cell profiles and spatial distributions. At P8, CSF interacts with microglia, vascular leptomeningeal cells (VLMCs), arachnoid barrier cells, oligodendrocyte precursor cells (OPCs), glial cells and spatially distinct neuronal populations including actively proliferating neural progenitor cells from the cerebellum, neuroblasts from the rostral migratory stream, and immature neurons from hippocampus and pontine nuclei. By P21, while CSF interacts with glial cell types at greater proportion, its interactions with neuronal populations, OPCs and VLMCs are significantly reduced. CellChat analysis reveals significant crosstalk among neuroglial populations, CNS vasculature, and microglia via ligand-receptor pathways linked to neurodegenerative diseases (PTN, MK) at both P8 and P21, and to neuronal migration (L1CAM, RELN) at P8.

Conclusion : These findings enhance our understanding of normal brain development and suggest potential intersections with the pathogenesis of brain fluid diseases such as hydrocephalus and neurodevelopmental disorders.