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

脊椎动物心脏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内皮祖细胞的增殖和通过强烈影响心脏瓣膜的形态发生而成为这些过程的重要触发因素。然而，一个全面的了解的机制，内皮细胞呈现敏感的流体流动剪切力和这些生物力学刺激如何触发内皮细胞的生长和形态缺乏。甚至更少的是已知的模式，其中流体流动剪切力对内皮细胞的机械影响是集成的，并在心脏形态发生过程中传输到相邻的心肌细胞。在这里，我们建议将联合收割机心脏组织RNA-seq分析与斑马鱼中细胞分辨率的心脏发育的体内成像相结合。我们最近制定了斑马鱼胚胎心脏瓣膜形态发生体内成像的条件，并建立了Tomo-seq协议，该协议提供了全转录组数据的高空间分辨率。结合斑马鱼胚胎心脏的光片成像，破译心脏基因表达将有助于识别心脏祖细胞的谱系关系，并解开瓣膜发生过程中单细胞行为的分子编排。我们的目标是解决的一个假设是，瓣膜形成是否是基于保守的endoMT过程的心脏祖细胞的问题，如果是这样，以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和内皮细胞表现出不同的反应，这些线索，我们还提出了不同的内皮细胞群体的存在与不同的阈值mechanosensitivity。我们的目标是通过在单细胞水平上解剖内皮细胞的分子特征并依赖于生物力学力来测试这一假设。最后，将利用单细胞数据来探测体内不同的细胞群，并在限制或允许形成心脏瓣膜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.