Sara Thornby Bak, Eva Bang Harvald, Ditte Gry Ellman, Sabrina Bech Mathiesen, Ting Chen, Shu Fang, Kristian Skriver Andersen, Christina Dühring Fenger, Mark Burton, Mads Thomassen, Ditte Caroline Andersen
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To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. 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引用次数: 2
摘要
胎儿心脏中的心肌细胞(CMs)会分裂,而出生后的 CMs 则无法进行核分裂和/或细胞分裂,因此会变成多倍体或双核,这是 CM 终末分化的一个关键过程。这种从二倍体增殖 CM 到终末分化的多倍体 CM 的转变仍是一个谜,似乎是心脏再生的一个障碍。在这里,我们利用单细胞 RNA 测序(scRNA-seq)来确定出生前后 CM 的转录格局,从而预测参与 CM 增殖和终末分化的转录因子(TFs)。为此,我们建立了一种方法,将荧光激活细胞分选(FACS)与来自发育中(E16.5、P1 和 P5)小鼠心脏的固定 CM 的 scRNA-seq 结合起来,生成了体内二倍体和四倍体 CM 的高分辨率单细胞转录组图,提高了 CM 的分辨率。我们发现了调控出生前后发育中CM的G2/M阶段的TF网络。ZEB1(Zinc Finger E-Box Binding Homeobox 1,锌指E-Box结合同源框1)是迄今为止在CM细胞周期中未知的TF,它被发现在E16.5时调控循环CM中数量最多的细胞周期基因,但在出生前后被下调。CM ZEB1-knockdown减少了E16.5 CM的增殖,而ZEB1在出生后P0期的过表达则导致CM的内复制。因此,这些数据提供了发育中CM的倍性分层转录组图谱,并为CM增殖和内再复制带来了新的见解,确定了ZEB1是这些过程中的关键角色。
Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth.
Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid proliferative CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes.
期刊介绍:
Basic Research in Cardiology is an international journal for cardiovascular research. It provides a forum for original and review articles related to experimental cardiology that meet its stringent scientific standards.
Basic Research in Cardiology regularly receives articles from the fields of
- Molecular and Cellular Biology
- Biochemistry
- Biophysics
- Pharmacology
- Physiology and Pathology
- Clinical Cardiology