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. 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.

脑周细胞（brain pericytes）对调控内皮屏障功能与活性至关重要，借此保障大脑的充足血液灌注。目前学界对发育过程中周细胞如何定植于裸露内皮的调控机制尚不完全明晰，但该机制与血管稳定性不足相关疾病密切相关。尽管周细胞可源自神经嵴与中胚层，但目前无法区分两种起源的脑周细胞；调控二者趋同形成周细胞表型的遗传通路仍未明确。本研究证实，同时源自神经嵴细胞（neural crest cell, NCC）与中胚层、表达转录因子nkx3.1的前体细胞群，可分化为脑周细胞。本研究鉴定出这类前体细胞的基因特征，并证实表达nkx3.1、foxf2a与cxcl12b的周细胞前体细胞群存在于表达cxcr4的基底动脉周围，且该细胞群后续会扩散至全脑。Cxcl12b-Cxcr4信号通路对周细胞的黏附与分化至关重要，但不参与周细胞后续的发育过程。此外，nkx3.1与cxcl12b均可调控周细胞数量，且二者均为该过程所必需。综上，本研究界定了一种尚未被报道的周细胞前体细胞群，并鉴定出对其分化至关重要的基因。将表达nkx3.1NTR-mcherry转基因的野生型30受精后小时（hours post fertilization, hpf）斑马鱼胚胎解离，对其中mCherry阳性的荧光激活细胞分选（fluorescence-activated cell sorting, FACS）所得细胞进行单细胞RNA测序。从FACS分选得到的表达nkx3.1的细胞中构建了两份重复测序文库。为探究特定细胞亚群的分子变化，我们从两份样本中分离单细胞，并按照10x基因组学（10X Genomics）Chromium单细胞3'端试剂试剂盒v3化学试剂盒的厂商操作规程进行处理。简言之，我们通过前向散射光与侧向散射光设门分选单细胞，将其收集于0.1%牛血清白蛋白-磷酸盐缓冲液（0.1% BSA-PBS）中，并利用10x GemCode™ 技术将单细胞封装于凝胶珠微滴（Gel Bead-In-EMulsions, GEMs）内。使用Illumina NovaSeq S2测序流动槽进行下一代测序。所有原始FASTQ文件均通过cellranger mkref流程构建的人类参考基因组进行序列比对。所得的基因条形码矩阵通过Seurat R工具包进行后续分析处理。