High throughput expression-based phenotyping and RNAi screening reveals novel regulators of planarian stem cells

The complexity of cell types and states revealed by scRNAseq cell atlases presents a challenge for the systematic analysis of fate determinants using traditional screening methodologies. Differentiation in the planarian Schmidtea mediterranea exemplifies this problem, as these animals continuously produce over 100 differentiated cell types for homeostasis and regeneration using adult pluripotent stem cells termed neoblasts. The signaling factors enabling neoblast self-renewal and selective differentiation of these many fates are still incompletely understood. We developed a method using high-throughput expression profiling by qPCR and whole-animal RNAseq to simultaneously assess numerous cell fate markers as the phenotypic readout in large-scale RNAi screens. Applying this method, we performed an RNAi screen of 400 kinases, receptors, and other regulatory molecules to reveal specific functions for 30 previously unknown factors in neoblast biology. 17 genes were required for neoblast maintenance, including factors likely involved in cell-cycle regulation, nutrient sensing, and chromatin modification. Multidimensional expression information additionally revealed several specific regulators of other neoblast activities, including a mink1 kinase regulating global neoblast differentiation, the energy responsive kinase adenylate kinase-2 regulating intestine specification within the neoblast population, an RNA acetyl transferase nat10 regulating epidermal differentiation, and a pak1 kinase that restricts neoblast localization to prevent tissue outgrowths. These results identify several new regulators of neoblast activities and demonstrate the applicability of expression-based screening for systematic analysis of stem cell phenotypes in whole animals. Total RNA was isolated from whole planarian animals after 21 days following RNAi of a control gene (Photinus pyralis luciferase, Ppluc), PAPSS2,CSNK1A1,ANK1,MINK1,PRPF4B,piwi-2,PANK3,LPT,MNAT1,Rb,nat10,CDK7,AK2,ANAPC,STK39,PLK1,TRRAP,LAMTOR,PKN2,AKT,MARK2,repA1,zfp-1, and bruli. 3 biological replicates of 4 animals each were analyzed for each condition. Worms were fed dsRNA every 2-3 days for 2 weeks and RNA harvested 7 days after the last feeding.

单细胞RNA测序（scRNAseq）细胞图谱所揭示的细胞类型与状态复杂性，给利用传统筛选方法系统性分析命运决定因子带来了挑战。地中海施密特涡虫（Schmidtea mediterranea）的分化过程便是这一问题的典型例证：这类动物依靠被称为新胚芽细胞（neoblasts）的成体多能干细胞，持续产生百余种分化细胞类型以维持稳态与再生能力。目前，介导新胚芽细胞自我更新以及选择性分化为众多细胞命运的信号因子仍未完全明确。
我们开发了一种结合实时定量PCR（qPCR）高通量表达谱分析与全动物RNA测序的方法，可在大规模RNA干扰（RNAi）筛选中同时以众多细胞命运标志物作为表型检测指标。
应用该方法，我们针对400种激酶、受体及其他调控分子开展了RNAi筛选，揭示了30个此前未被报道的新胚芽细胞生物学相关因子的特定功能。其中17个基因对新胚芽细胞的维持不可或缺，涵盖可能参与细胞周期调控、营养感应及染色质修饰的各类因子。多维表达分析还进一步揭示了数种调控新胚芽细胞其他活动的特异性调控因子：包括mink1激酶可调控新胚芽细胞的整体分化进程；能量响应激酶腺苷酸激酶2（adenylate kinase-2）可调控新胚芽细胞群体内的肠道特化过程；RNA乙酰转移酶nat10可调控表皮分化；而pak1激酶则可限制新胚芽细胞的异常定位，以防止组织过度增生。
本研究不仅鉴定出多种调控新胚芽细胞活动的新型调控因子，同时证明了基于表达谱的筛选方法可用于系统性分析整只动物体内的干细胞表型。
我们从经RNAi处理21天后的整只涡虫体内提取总RNA，所靶向的基因包括阴性对照基因（萤火虫荧光素酶Photinus pyralis luciferase，简称Ppluc）以及PAPSS2、CSNK1A1、ANK1、MINK1、PRPF4B、piwi-2、PANK3、LPT、MNAT1、Rb、nat10、CDK7、AK2、ANAPC、STK39、PLK1、TRRAP、LAMTOR、PKN2、AKT、MARK2、repA1、zfp-1与bruli。
每组实验设置3个生物学重复，每个重复包含4只涡虫。
实验中每2-3天给涡虫喂食双链RNA（dsRNA），持续2周，并在最后一次喂食7天后收集样本提取RNA。