Developmental heterogeneity of embryonic neuroendocrine chromaffin cells and their maturation dynamics. Developmental heterogeneity of embryonic neuroendocrine chromaffin cells and their maturation dynamics

During embryonic development, nerve-associated Schwann cell precursors (SCPs) give rise to chromaffin cells of the adrenal gland via the “bridge” transient stage, according to recent functional experiments and single cell data from humans and mice. However, currently existing data do not resolve the finest heterogeneity of developing chromaffin populations. Here we took advantage of deep SmartSeq2 transcriptomic sequencing to expand our collection of individual cells from developing murine sympatho-adrenal anlage and uncover the microheterogeneity of embryonic chromaffin cells and corresponding developmental paths. After improving our atlas of sympatho-adrenal development and performing experimental validations, we discovered that SCPs in the local nerve show high degree of microheterogeneity corresponding to early biases towards either Schwann or chromaffin terminal fates. Furthermore, we found that a post-“bridge” population of developing chromaffin cells gives rise to persisting immature chromaffin cells and the two terminal populations (adrenergic and noradrenergic) via diverging differentiation paths. Taken together, we provide a thorough identification of novel markers of adrenergic and noradrenergic populations in developing adrenal glands and report novel differentiation micro-paths leading to them. Overall design: Smart-Seq2 protocol was performed on single isolated cells by Eukaryotic Single Cell Genomics Facility at SciLifeLab, Stockholm. Single TOMATO+ cells were sorted into 384-well plates prefilled with lysis buffer according to the previously published SmartSeq2 protocol (Picelli, Faridani et al. 2014) using a BD FACSAria Fusion Cell Sorter B5/R3/V3 system with a three-laser configuration (488nm, 633nm, and 405nm) and 16 fluorescence detectors. Single-cell transcriptomic sequencing was performed as previously described (Picelli, Faridani et al. 2014).

现有针对人类与小鼠的功能实验及单细胞数据表明，在胚胎发育阶段，神经伴随的施万细胞前体（Schwann cell precursors, SCPs）可通过“桥接”过渡阶段分化为肾上腺嗜铬细胞（chromaffin cells）。然而，当前已有的数据尚无法解析发育中嗜铬细胞群体最精细的异质性。本研究借助深度SmartSeq2转录组测序技术，扩充了发育中小鼠交感肾上腺原基（sympatho-adrenal anlage）的单细胞样本库，并解析了胚胎嗜铬细胞及其对应发育轨迹的微异质性。在优化我们的交感肾上腺发育图谱并完成实验验证后，本研究发现局部神经中的施万细胞前体（SCPs）存在高度的微异质性，对应其早期偏向施万细胞或嗜铬细胞终末命运的分化倾向。此外，本研究发现桥接阶段后的发育中嗜铬细胞群体，可通过分化轨迹分支产生持续存在的未成熟嗜铬细胞以及两类终末群体（肾上腺素能与去甲肾上腺素能嗜铬细胞）。综上，本研究全面鉴定了发育中肾上腺内肾上腺素能与去甲肾上腺素能细胞群体的新型标志物，并报道了指向这些群体的新型分化微路径。整体实验设计：本研究由斯德哥尔摩瑞典生命科学实验室（SciLifeLab）的真核单细胞基因组学平台完成单细胞Smart-Seq2实验。依照已发表的SmartSeq2实验方案（Picelli、Faridani等，2014），使用搭载三激光配置（488nm、633nm与405nm）及16个荧光检测器的BD FACSAria Fusion细胞分选仪B5/R3/V3系统，将单阳性TOMATO+细胞分选至预先添加了裂解缓冲液的384孔板中。单细胞转录组测序流程依照此前报道的方法（Picelli、Faridani等，2014）完成。