Digital and molecular reconstruction of zebrafish heart morphogenesis by single-cell transcriptomics and light-sheet microscopy

脊椎动物心脏的发育依赖于控制祖细胞群的产生和分配并指导形态发生的分子途径。由血流和心脏收缩力施加的机械力通过影响心内膜祖细胞的增殖和强烈影响心脏瓣膜的形态发生，是这些过程的重要触发因素。然而，对于心内膜细胞对流体剪切力敏感的机制，以及这些生物力学刺激如何触发心内膜生长和形态发生，目前还缺乏全面的了解。在心脏形态发生过程中，流体流动剪切力对心内膜细胞的机械影响是如何整合并传递给邻近心肌细胞的，我们对这种模式的了解就更少了。在这里，我们建议将心脏组织RNA-seq分析与斑马鱼细胞分辨率下心脏发育的体内成像相结合。我们最近研究出了斑马鱼胚胎心脏瓣膜形态发生的体内成像条件，并建立了Tomo-seq协议，该协议提供了高空间分辨率的全转录组数据。结合斑马鱼胚胎心脏的光片成像来破译心脏基因表达将有助于确定心脏祖细胞的谱系关系，并揭示瓣膜发生过程中单细胞行为的分子协调。我们旨在解决的一个假设是瓣膜发生是否基于心脏祖细胞保守的endoMT过程，如果是这样，阐明生物力学刺激如何影响这些过程的调节。由于生物力学力在心脏的不同区域不同，心内膜细胞对这些信号表现出不同的反应，我们还提出存在不同的心内膜种群，具有不同的机械敏感性阈值。我们的目标是通过在单细胞水平上解剖心内膜的分子特征和生物力学力的依赖性来检验这一假设。最后，将利用单细胞数据来探测体内不同的细胞群，并在限制或允许心脏瓣膜形成的条件下使用实时成像来研究它们的行为。

Vertebrate cardiac development depends on molecular pathways that control the generation and allocation of progenitor cell populations and guide morphogenesis. Mechanical forces exerted by blood flow and cardiac contractility are important triggers of these processes by impacting the proliferation of endocardial progenitor cells and by strongly affecting the morphogenesis of heart valves. However, a comprehensive understanding of the mechanisms by which endocardial cells are rendered sensitive to fluid flow shear forces and how these biomechanical stimuli trigger endocardial growth and morphogenesis is lacking. Even less is known about the modes by which the mechanical impact of fluid flow shear forces on endocardial cells is integrated and transmitted to neighboring myocardial cells during cardiac morphogenesis. Here, we propose to combine cardiac tissue RNA-seq analyses with in vivo imaging of cardiac development at a cellular resolution in zebrafish. We have recently worked out conditions for in vivo imaging of heart valve morphogenesis in zebrafish embryos and established a protocol for Tomo-seq, which provides high spatial resolution of whole-transcriptome data. Deciphering cardiac gene expression in combination with light-sheet based imaging of zebrafish embryonic hearts will help to identify lineage relationships of cardiac progenitor cells and unravel the molecular orchestration of single cell behaviors during valvulogenesis. One hypothesis that we aim to address is the question whether valvulogenesis is based on conserved endoMT processes of cardiac progenitor cells and, if so, to elucidate how biomechanical stimuli impact the regulation of these processes. Since biomechanical forces vary in different regions of the heart and endocardial cells show different responsiveness to these cues, we also propose the existence of distinct endocardial populations with varying thresholds of mechanosensitivity. We aim to test this hypothesis by dissecting the molecular signature of the endocardium at a single cell level and in dependence of biomechanical forces. Finally, single cell data will be utilized to probe for distinct cell populations in vivo and their behaviors will be studied using live imaging under conditions that are restrictive or permissive for the formation of cardiac valves.