The ECM as a coordinator of cardiac function, growth and morphogenesis during heart development

心脏是一个复杂的器官，它的形状与功能紧密相连。在胚胎发育过程中，心脏最初是一个线形管，经过重排形成其最终形态。这个过程被称为心脏形态发生，如果发生不当，可能导致出生异常，即先天性心脏缺陷。当心脏正在进行形态发生时，还会发生两个对心脏发育至关重要的事件。首先，心脏的大小是通过向心脏两极添加来自周围中胚层的细胞祖池的细胞来增长的，这些细胞被称为第二心脏场（SHF）。虽然向心脏添加SHF细胞是心脏形态发生的重要组成部分，但我们对这些细胞如何融入心脏知之甚少。其次，心脏在形态形成时已经在跳动。我们之前的工作表明，心脏收缩控制着SHF细胞如何添加到心脏中，并将心脏生长与形态发生结合起来。细胞和组织不是孤立存在的——细胞周围的环境是一种叫做细胞外基质（ECM）的蛋白质和糖分子的多样化组合，其组成对不同的组织是特定的。ECM调节向周围细胞传递的生化信号，提供机械支持和生物力学线索，是细胞迁移、组织形态发生和器官功能的关键调节剂。因此，ECM是心管和周围SHF的重要组成部分。我们已经证明，ECM成分层粘连蛋白可能通过调节SHF细胞的收缩影响来调节SHF的添加，从而在心脏功能和生长之间提供了新的联系。然而，心功能、ECM和SHF添加在耦合生长和形态发生中的关系尚不清楚。在这个建议中，我们使用斑马鱼胚胎心脏发育的实时成像，在心脏功能丧失或增加的情况下，了解收缩力和ECM在调节SHF添加方面的关系。斑马鱼代表了一种独特的模式生物，使我们能够定义这种关系。胚胎是透明的，在外部发育，所以我们可以看到一个活胚胎的心脏发育。此外，斑马鱼可以在没有心脏功能的情况下存活几天，这意味着我们可以研究心脏功能如何影响心脏发育，这在其他生物中是具有挑战性的。我们将通过三个目标来定义这种关系，所有这些目标都将利用这种能力对发育中的斑马鱼心脏和SHF进行活体成像。首先，我们将停止或加速胚胎斑马鱼的心脏，并评估收缩性如何影响ECM本身。我们将在心脏形态发生期间可视化ECM的数量和分布，并将标记特定的ECM成分，以可视化收缩性改变后的ECM组织和组成。其次，我们将通过对跳动的心管和随时间迁移的SHF细胞进行成像，确定ECM成分层粘连蛋白的缺失如何影响心管收缩性和SHF细胞的运动。第三，我们将结合前两个目标的实验方法来定义收缩力和/或ECM的变化如何共同影响心脏生长速度。我们将在目标1和目标2的实验模型中生成发育中的心脏的最先进的3D重建，使我们能够首次定义收缩性和ECM如何通过中介生长共同塑造心脏。定义ECM如何联系心脏功能、生长和形态有许多应用。这将有助于理解驱动人类心脏发育的过程，以及ECM在发育过程中如何塑造身体其他组织。ECM最近也被确定为心脏再生的关键驱动因素，因此这项工作对提高我们改善损伤后组织再生的能力具有重要意义。

The heart is a complex organ whose shape is tightly linked to function. During embryonic development the heart is initially a linear tube which undergoes rearrangement to generate its final form. This process is called heart morphogenesis, and if it does not occur properly can lead to birth abnormalities known as congenital heart defects. While the heart is undergoing morphogenesis, two additional events also occur which are crucial for heart development. First, the heart grows in size through the addition of cells to the poles of the heart from a progenitor pool of cells in the surrounding mesoderm called the Second Heart Field (SHF). Although the addition of SHF cells to the heart is an essential part of heart morphogenesis, we understand very little about how these cells become incorporated into the heart. Second, the heart is already beating during morphogenesis. Our previous work suggests that heart contraction controls how SHF cells are added to the heart, and couples heart growth with morphogenesis.Cells and tissues do not exist in isolation - the environment around cells is a diverse assortment of protein and sugar molecules called the extracellular matrix (ECM), the composition of which is specific to different tissues. The ECM regulates biochemical signalling to surrounding cells, provides mechanical support and biomechanical cues, and is a key regulator of cell migration, tissue morphogenesis, and organ function. The ECM is therefore a crucial component of the heart tube and surrounding SHF. We have shown that the ECM component laminin regulates SHF addition, likely by regulating the impact of contractility on SHF cells, providing a new link between heart function and growth. However, the relationship between cardiac function, the ECM, and SHF addition in coupling growth and morphogenesis is unknown.In this proposal we use live imaging of heart development in zebrafish embryos where heart function is lost or increased to understand the relationship between contractility and the ECM in regulating SHF addition. Zebrafish represent a unique model organism allowing us to define this relationship. The embryos are transparent and develop externally, so we can visualise heart development in a live embryo. Furthermore, zebrafish can survive the first few days of development without a functioning heart, meaning we can investigate how heart function impacts heart development in a way that is challenging in other organisms.We will define this relationship via three objectives, all of which will exploit this ability to perform live in vivo imaging of the developing zebrafish heart and SHF. First, we will stop or speed up the embryonic zebrafish heart and assess how contractility affects the ECM itself. We will visualise the amount and distribution of ECM in the heart during morphogenesis, and will label specific ECM components to visualise ECM organisation and composition upon altered contractility. Second, we will define how loss of the ECM component laminin affects heart tube contractility and SHF cell movement by imaging the beating heart tube and migrating SHF cells over time. Third we will combine the experimental approaches from the first two objectives to define how changes in contractility and/or the ECM together affect the rate of heart growth. We will generate state-of-the-art 3D reconstructions of the developing heart in the experimental models from objectives 1 and 2, enabling us to define for the first time how contractility and the ECM together shape the heart by mediating growth. Defining how the ECM links heart function, growth, and morphology has many applications. It will help understand the processes driving heart development in humans, as well as how the ECM shapes other tissues in the body during development. The ECM has also been recently identified as a key driver of heart regeneration, so this work has important implications in advancing our ability to improve tissue regeneration after injury.