scRNAseq datasets supporting a common precursor expression program leading to brain pericyte differentiation from neural crest and mesoderm

Brain pericytes are critical for regulating endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. How the developmental acquisition of pericytes to naked endothelium is regulated, is largely unknown, but is relevant to disorders where vessels are poorly stabilized. Although pericytes are derived from neural crest and mesoderm, brain pericytes from both origins are currently indistinguishable; the genetic pathways leading to a convergent pericyte phenotype are unknown. We show here that a precursor population expressing the transcription factor nkx3.1 with origins in both NCC and mesoderm, develops into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1, foxf2a, and cxcl12b -expressing pericyte precursor population is present around the cxcr4 expressing basilar artery, and that these cells later spread throughout the brain. Cxcl12b- Cxcr4 signaling is required for pericyte attachment and differentiation but not for later pericyte development. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number. Thus, we have defined an undescribed population of pericyte precursors and identified genes critical for their differentiation. Overall design: Embryos expressing the nkx3.1NTR-mcherry transgene were dissociated from wildtype 30 hpf zebrafish embryos and mCherry-positive FACs sorted cells were subjected to single cell RNA sequencing. Two replicate libraries were generated from FACS-sorted nkx3.1 expressing cells . To examine molecular changes within specific cell compartments, we isolated single-cells from both biopsies and processed according to 10X Genomics Chromium Single Cell 3' Reagent Guidelines v3 Chemistry as per the manufacturer's protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq S2 Flow cells. All raw FASTQs were aligned to the human reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix was processed using Seurat R toolkit.