Single-nucleus resolution of the adult C. elegans and its application to the mapping of inter-generational response to alcohol exposure [scRNA-seq]

Single-cell RNA sequencing technologies have significantly contributed to our understanding of tissue heterogeneity as well as developmental and cell differentiation trajectories. They also provide an opportunity to map an organism’s response to environmental cues with high resolution and identify sensitive tissues and cell types. However, the requirement for cellular integrity during extraction has limited the examination of organs that are syncytial or of cell types, such as neurons, that have significant cellular projections. We developed a mechanical method for single nucleus extraction in C. elegans that, combined with snRNA-seq, identifies a remarkable number of cell types in the adult nematode. Clustering analysis recapitulated established morphological and functional categories as well as the differentiation trajectory of the syncytial germline. Our approach also uncovered novel features of C. elegans biology, such as previously uncharacterized intestinal subpopulations and the up-regulation of X-linked transcripts in the somatic compartment of the hermaphrodite spermatheca. We applied this method to identify the impact of intergenerational exposure to a human-relevant dose of alcohol. We show that parental ethanol exposure causes a highly cell type-specific response in the adult F1 with oocytes, motor neurons, mechanosensory neurons, and neuronal support cells displaying the highest number of differentially expressed genes, the majority of which were downregulated. Cluster-level GO analysis revealed pathways previously not known to be altered by ethanol such as sister chromatid dynamics in the oocyte and autophagy in the uterine epithelium. Together, snRNA-seq provides a powerful approach for the detailed examination of an adult organism physiology. 31 single nuclei C. elegans datasets were generated, divided into 5 batchs, four treatment group (normal, water, 0.5% ethanol, 0.05% ethanol) and two generations (F1 and F3)

单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术极大地推动了我们对组织异质性、发育及细胞分化轨迹的认知，同时为高分辨率解析生物体对环境刺激的应答、鉴定敏感组织与细胞类型提供了可能。然而，样本提取过程中对细胞完整性的要求，限制了对合胞体结构器官或具有显著细胞突起的细胞类型（如神经元）的研究。本研究开发了一种用于秀丽隐杆线虫（Caenorhabditis elegans, C. elegans）单细胞核提取的机械方法，结合单细胞核RNA测序（single nuclear RNA sequencing, snRNA-seq）技术，可鉴定成年线虫中极为丰富的细胞类型。聚类分析重现了已确立的形态学与功能分类，以及合胞体生殖系的分化轨迹。本方法还揭示了秀丽隐杆线虫生物学的全新特征，例如此前未被表征的肠道亚群，以及雌雄同体受精囊体细胞区域内X连锁转录本的上调现象。我们将该方法应用于鉴定跨代暴露于人体相关剂量酒精的影响，研究表明，亲代乙醇暴露会在成年F1代中引发高度细胞类型特异性的应答：卵母细胞、运动神经元、机械感觉神经元以及神经元支持细胞的差异表达基因数量最多，且其中绝大多数呈现下调趋势。基于聚类的基因本体（Gene Ontology, GO）富集分析揭示了此前未被报道受乙醇调控的通路，例如卵母细胞中的姐妹染色单体动态变化以及子宫上皮细胞中的自噬过程。综上，单细胞核RNA测序技术为详细解析成体生物体生理状态提供了强有力的研究手段。本研究共生成31个秀丽隐杆线虫单细胞核数据集，分为5个批次，涵盖4个处理组（正常对照组、纯水对照组、0.5%乙醇组、0.05%乙醇组）以及2个世代（F1代与F3代）