Project summary The meninges encase the CNS from its earliest stages of development and persist as a protective covering for the adult CNS. Many studies show the meninges, in particular the meningeal fibroblasts, has vital roles in controlling developmental neurogenesis and neuronal migration and congenital defects in human brain development can arise from meningeal defects. The meninges also contain unique immune and vascular cell populations (blood and lymphatics) that are now known to serve key functions in neuro-immune surveillance, cerebrospinal fluid drainage and CNS waste removal, thus supporting overall CNS health and function. Despite this, we have an incomplete understanding of the cellular and molecular mechanisms that control formation of the meninges. We know very little about the development of the different fibroblast populations that produce factors that help control brain development and may have roles in development of other meninges cell types (immune cells) and adjacent structures like the calvarium. Our prior work demonstrates that meningeal fibroblast populations of the pia, arachnoid and dura layers around the forebrain are transcriptionally unique and begin to express layer specific markers at early stages of brain development. We and others have previously shown that the transcription factor Foxc1 is key for this meningeal layer specification. Here we propose to use a new, meninges conditional Foxc1 mouse mutant in combination with single cell transcriptomics (scRNAseq) to begin to identify: 1) the normal developmental progression of meningeal fibroblast layer populations and identify signaling pathways that underlie the failed development in Foxc1 mutants and 2) identify signals produced by prenatal meningeal fibroblasts that control maturation of other meningeal populations, in particular immune cells, and the adjacent calvarium. Completion of experiments outlined here will serve as foundational pilot data for future grant applications, support our long- term research goals in studying meninges control of CNS development and function and will be an important resource for researchers studying meninges-related structure, process, disease or malformation.