Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Schmidtea mediterranea

Flatworms of the species Schmidtea mediterranea are immortal –adult animals contain a large pool of pluripotent stem cells that continuously differentiate to all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. Here, by combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from the transcriptional changes, and a novel lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterize gene expression changes during differentiation and discover cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at unprecedented resolution. Overall design: Single cell sequencing by drop-seq of Schmidtea mediterranea whole cells including 2 bulk population wild type samples fixed in methanol, 1 bulk population wild type sample cryopreserved, 2 bulk population wild type alive sample and 2 bulk population after control and Smed-h2b RNAi samples fixed in methanol and 3 wt samples at 3 different regeneration time points (day 0, day 2, day 4) fixed in methanol.

地中海真涡虫（Schmidtea mediterranea）具有永生特性——成体个体拥有大量多能干细胞池，这些干细胞可持续分化为所有成体细胞类型。因此，对成体涡虫开展单细胞转录组谱分析（single-cell transcriptome profiling），可揭示成熟细胞与祖细胞的特征。本研究结合扰动实验、基因表达分析、一种基于转录变化预测细胞未来状态的计算方法，以及一种全新的谱系重构方法，将所有主要细胞类型整合至单一谱系树中，该谱系树可将所有细胞溯源至单个干细胞区室。本研究刻画了分化过程中的基因表达变化，并发现了对再生至关重要的细胞类型。本研究结果证实，单细胞转录组谱分析能够以空前的分辨率，绘制并重构发育生物学与再生生物学的核心生理过程。实验设计概述：采用液滴单细胞测序（drop-seq）技术对地中海真涡虫的全细胞进行测序，所涉样本包括：2份甲醇固定的野生型混合细胞群体样本、1份冷冻保存的野生型混合细胞群体样本、2份活体野生型混合细胞群体样本、2份分别经对照及Smed-h2b RNA干扰（RNAi）处理后甲醇固定的混合细胞群体样本，以及3份处于3个不同再生时间点（第0天、第2天、第4天）的甲醇固定野生型样本。