Patterning mechanisms that restrict cardiac cushion formation in zebrafish

描述（申请人提供）：心脏瓣膜通过促进单向血液流动在心脏功能中发挥关键作用，是先天性心脏病的常见目标。房室瓣膜的形成是由精确定位于室间连接蛋白（也称为房室管或AVC）的信号触发的。这些信号指示局部的心内膜膨胀到心管的腔内，形成称为心垫的结构，然后再改造成瓣膜小叶。斑马鱼的研究表明，典型的Wnt通路在AVC的活性促进编码Bmp信号的基因的表达，从而诱导缓冲层的形成。正常的心脏发育需要限制这些感应信号到AVC，但我们不了解这种限制的机制。我们也不知道这个系统的重要一般属性，比如Wnt信号是否自己模式，或者它是否中继上游模式。我将追求两个具体目标来扩展我们对AVC模式网络的组件和系统级属性的知识。我的第一个目标是研究血清素信号，我们的初步数据表明它在AVC模式中起着关键作用。用5 -羟色胺受体拮抗剂酮色胺处理胚胎导致bmp4异位表达超出AVC边界。这种表型通过5 -羟色胺受体htr2a的敲低重现，表明5 -羟色胺通过该受体的信号传导是必不可少的。我假设血清素信号参与了缓冲诱导信号对AVC的限制。为了验证这一假设，我将首先检查一组标记，以彻底表征htr2a缺陷胚胎中的缓冲模式缺陷。接下来，我将使用药理学扰动来确定血清素信号对AVC模式至关重要的时间窗口。我还将检查Wnt通路的读数，并进行上位实验，以确定血清素是否在Wnt通路的上游或下游起作用，以限制缓冲诱导。最后，我将使用全球血清素水平的药理学扰动结合htr2a错误表达来确定血清素信号是指导还是允许缓冲限制。在我的第二个目标中，我将测试Wnt信号模式本身的假设，即自组织。自组织系统具有一些有用的性质，如稳定性和对波动的鲁棒性。这些系统通常包含一个自我放大成分（正反馈），通过自身（自抑制）或竞争对手的自我放大产生的抑制来平衡

DESCRIPTION (provided by applicant): Heart valves play a critical role in cardiac function by promoting unidirectional blood flow and are a frequent target of congenital heart disease. Atrioventricular valve formation is triggered by signals that are precisely localized to the junctin between chambers (also known as the atrioventricular canal or AVC). These signals instruct the local endocardium to bulge into the lumen of the heart tube, forming structures known as cardiac cushions that later remodel into valve leaflets. Studies in zebrafish have shown that canonical Wnt pathway activity at the AVC promotes the expression of genes encoding Bmp signals that induce cushion formation. Proper heart development necessitates the restriction of these inductive signals to the AVC, yet we do not understand the mechanisms responsible for this confinement. We also do not know important general properties of this system, such as whether Wnt signaling patterns itself or if it relays an upstream pattern. I will pursue two specifc aims to expand our knowledge of the components and systems-level properties of the AVC patterning network. My first aim investigates serotonin signaling, which our preliminary data suggest has a critical role in AVC patterning. Treatment of embryos with the serotonin receptor antagonist ketanserin results in ectopic bmp4 expression beyond the boundaries of the AVC. This phenotype is recapitulated by knockdown of the serotonin receptor htr2a, suggesting that serotonin signaling through this receptor is essential. I hypothesize that serotonin signaling participates in the confinement of cushion-inducing signals to the AVC. To test this hypothesis, I will first examine a panel of markers to thoroughly characterize the cushion patterning defect in htr2a-deficient embryos. I will next use pharmacological perturbations to determine the time window when serotonin signaling is critical for AVC patterning. I will also examine Wnt pathway readouts and perform epistasis experiments to determine whether serotonin acts upstream or downstream of the Wnt pathway to restrict cushion induction. Finally, I will use pharmacological perturbations of global serotonin levels in combination with htr2a misexpression to determine whether serotonin signaling is instructive or permissive for cushion restriction. In my second aim, I will test the hypothesis that Wnt signaling patterns itself, i.e. self- organizes. Self-organizing systems have several useful properties such as stability and robustness to fluctuations. These systems often contain a self-amplifying component (positive feedback) that is balanced by inhibition generated either by itself (autoinhibition) or by a rival self-amplifying component that dominates an adjacent domain (inhibitory crosstalk). I will use a combination of novel optogenetic techniques and live signaling reporters to determine whether Wnt signaling in the AVC displays hallmark characteristics of self-organizing systems, such as positive feedback, autoinhibition, and inhibitory crosstalk. Together, these studies will reveal important new mechanistic information and systems-level properties of the AVC patterning network, which will help us to understand the etiology of congenital heart defects.