Chromatin accessibility profiles 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 ATAC sequencing investigating chromatin accessibility profiles of cells harvested from control and Mesp1 KO whole embryos ages E7.5 and E7.75.

调控心脏前体细胞（cardiac precursor cell, CPC）特化的转录调控网络尚未被完全阐明，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细胞。我们借助小鼠胚胎高精度单细胞转录组时间序列中的早期心脏谱系转基因标记检测，成功鉴定出新生心脏前体细胞，并描述了其转录组特征。Mesp1作为一种瞬时表达的中胚层转录因子（transcription factor, TF），传统上被认为是心脏特化的早期调控因子。然而我们观察到，尽管定位异常，但Mesp1敲除（KO）小鼠中仍存在表达心脏前体细胞转基因标记的细胞，这促使我们探究Mesp1在心脏前体细胞出现与分化中的作用范围。Mesp1敲除的心脏前体细胞无法有效激活心肌细胞成熟标记及关键心脏转录因子，但其转录组特征却类似正向心肌细胞命运分化的心脏中胚层。单细胞染色质开放性分析明确了心脏谱系发育过程中一个依赖Mesp1的发育断点：该断点对应转录调控网络从内中胚层网络转向心脏模式形成与形态发生所需网络的过程。上述结果揭示了早期心脏前体细胞特化中不依赖Mesp1的途径，并强调了心脏发生过程中推进分化所需的依赖Mesp1的调控框架。实验设计：采用单细胞ATAC测序（ATAC sequencing），分析采集自发育阶段E7.5与E7.75的野生型对照及Mesp1敲除小鼠全胚胎细胞的染色质开放性谱图。