Single-cell RNA-seq profiling of early embryonic development in Ciona Robusta

During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process “transcriptome maturation”, and identified candidate “mature genes”, including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of “behavioral competence”. Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs. Overall design: Samples from 5-14hpf Ciona embryos were tagged with a pair of sample barcoding (SBC) constructs as unique labels for the multiplexing strategy. Singel-cell suspensions were prepared as described in the manuscripts. Whole embryo cells were used to generate single-cell libraries following a modified 10X protocol to capture the sequencing reads from both the transcriptome and SBC transcripts.

在个体发育过程中，干细胞与祖细胞会进行分裂，并依次经历胚层与谱系特异性的多能状态，最终分化构成动物体的各类细胞类型。生物分子组成的确定性变化，是多能性逐步丧失、细胞获得谱系特异性特征的分子基础。例如，多能性心咽祖细胞会呈现多谱系转录预激活状态：心肌与咽肌的发育程序会在同一祖细胞内同时处于部分激活的共存状态，而其子细胞仅在细胞分裂完成后，才会分别走向心肌或咽肌的分化路径。本研究以被囊动物玻璃海鞘（Ciona）为模型，探究了多谱系潜能的获得过程，以及细胞命运决定与细胞周期进程之间的耦合机制。我们证实，多能性心咽祖细胞会在有丝分裂前不久获得生成表型各异的Tbx1/10阳性（+）与阴性（-）子细胞的潜能，而这一过程是Tbx1/10基因激活的必要前提。本研究将基于转基因的样本条形码（sample barcoding, SBC）标记技术与单细胞RNA测序（single cell RNA-seq, scRNA-seq）相结合，揭示了迁移状态下心咽祖细胞在经历G1、S与G2期时的全转录组动态变化。我们将这一过程命名为"转录组成熟化"，并筛选出了一批候选"成熟基因"，其中包括在G2晚期表达达到峰值的Rho GTP酶激活蛋白（Rho GAP）编码基因Depdc1。功能实验表明，转录组成熟化通过多谱系预激活、调控细胞定向与不对称分裂等方式，促进心咽祖细胞的潜能获得，这一过程会影响后续的细胞命运决定，印证了"行为潜能"这一概念。经典前馈回路与细胞周期进程的耦合共同驱动转录组成熟化，由此揭示了细胞周期进程与命运决定相关分子转变之间的多层次耦合关系。我们提出，将潜能获得与命运决定分别与G2期和G1期进行耦合，能够确保谱系特异性发育程序的按时启动。实验整体设计：本研究对受精后5至14小时（5-14hpf）的玻璃海鞘胚胎样本，通过一对样本条形码（SBC）构建体进行标记，以此作为多重测序策略的唯一识别标签。按照本研究既定实验方案制备单细胞悬液，采用改良的10X Genomics建库流程，从全胚胎细胞中构建单细胞文库，以同时捕获转录组与样本条形码转录本的测序reads。