Transcriptional specification of early cardiac precursor cells at single cell resolution. Transcriptional specification of early cardiac precursor cells at single cell resolution

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood due in part to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently-expressed mesodermal transcription factor (TF), is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mis-localized, prompting us to investigate the scope of Mesp1’s role in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and critical cardiac TFs, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis. Overall design: Single cell RNA sequencing profiling transcriptional profiles of control and Mesp1 KO cells harvested from whole embryos in a time course of murine gastrulation stages E6.0-E7.75.

调控心脏前体细胞（cardiac precursor cell, CPC）命运特化的转录调控网络目前尚未被完全阐明，部分原因在于早期原肠胚形成阶段难以区分心脏前体细胞与非心脏中胚层。本研究借助小鼠胚胎精细单细胞转录组时间序列中早期心脏谱系转基因的检测手段，鉴定了新生的心脏前体细胞，并描述了其转录组特征。Mesp1是一种瞬时表达的中胚层转录因子（transcription factor, TF），经典上被认为是心脏特化的早期调控因子。然而我们观察到，在Mesp1突变体中，表达心脏前体细胞转基因的细胞仍持续存留，尽管其定位存在异常；这促使我们探究Mesp1在心脏前体细胞产生与分化过程中的作用范围。Mesp1突变的心脏前体细胞无法有效激活心肌细胞成熟标志物以及关键心脏转录因子，但其转录组特征却与向心肌细胞命运分化的心脏中胚层相似。单细胞染色质开放性分析显示，在心脏谱系发育从以中内胚层转录网络为主转向心脏模式形成与形态发生所需转录网络的过程中，存在一个Mesp1依赖的发育转折点。本研究结果揭示了早期心脏前体细胞特化中不依赖Mesp1的调控通路，并强调了心脏发生进程所需的Mesp1依赖的调控网络。实验设计概述：在小鼠原肠胚形成阶段E6.0至E7.75的时间序列中，收集完整胚胎中的野生型与Mesp1敲除细胞，通过单细胞RNA测序分析其转录组特征。