21 22 Key words (3-6 words): brain development, meninges, pial basement membrane, retinoic acid, human 23 meninges 24 2 Abstract 25The meninges, a multilayered structure that encases the CNS, is composed mostly of fibroblasts, 26 along with vascular and immune cells. Meningeal fibroblasts are a vital source of signals that control 27 neuronal migration and neurogenesis yet strikingly little is known about their development. We used 28 single cell RNA sequencing to generate a cellular atlas of embryonic meningeal fibroblasts in control and 29 Foxc1-KO mice in which severe CNS defects arise from failed meningeal fibroblast development. We 30 report unique transcriptional signatures for dura, arachnoid and pial fibroblasts and identify S100a6 as the 31 first unique marker of the pial layer. We describe a new meningeal fibroblast subtype marked by µ-32Crystallin expression and show these cell types and markers are conserved in human fetal meninges. Our 33 analysis demonstrates layer specific production of extracellular matrix components, transporter 34 expression, and synthesis of secreted factors. Lastly, the cellular atlas of Foxc1-KO meninges provides 35 insight into their severe phenotype, confirming a massive loss in arachnoid and dura fibroblasts and Foxc1-36 KO pial fibroblasts are so altered that they cluster as a different cell type based on gene expression. These 37 studies provide an unprecedented view of meningeal fibroblast development, highlighting unexpected 38 fibroblast diversity and function, while providing mechanistic insights into the meninges role in CNS 39 development. 40 41 42 4 of these specialized fibroblasts, mechanisms that guide their development, and their functions in the 68 developing and adult brain. 69Here we present single cell RNA sequencing (scRNA-seq) analysis of E14.5 mouse telencephalic 70 meningeal fibroblasts from control and Foxc1-KO mice. We define the transcriptional signatures of 71 embryonic pial, arachnoid and dural fibroblasts, including identification of a first-ever pial fibroblast 72 marker, S100a6. We present evidence for a previously unknown subtype of meningeal fibroblast we term 73 ceiling cells, so named as the meninges are like a 'ceiling' for the brain, that are enriched for expression 74 of µ-Crystallin. We show that our novel markers for the pia (S100a6), the arachnoid (CRABP2) and 75 ceiling cells (µ-Crystallin) in mouse identify specific meningeal populations in the human fetal brain, 76 indicating development of meningeal fibroblasts is conserved between mouse and human. ScRNA-seq 77 and cluster analysis of the Foxc1-KO meninges fibroblasts revealed a near absence of pial, arachnoid and 78 dural fibroblasts and, rather unexpectedly, a pial-like meningeal fibroblast population that clustered 79 separately in the single cell data analysis. We believe our single cell analyses of the embryonic meningeal 80 fibroblasts will provide a valuable resource that will aid in the study of embryonic and, possibly, the adult 81 meningeal fibroblast populations. 82Resul...