Wnt pathway dynamics and their role in generating self-organized ectodermal patterns

项目摘要 Wnt通路在胚胎发育和疾病中起着至关重要的作用。早期 发育，原肠胚形成和中内胚层分化需要Wnt信号传导，并且， 稍晚，用于外胚层内神经嵴细胞的分化。而 信号转导的详细生物化学已被广泛研究，但知之甚少 关于信号通路活动的动力学。我们使用CRISPR-Cas9基因组工程， 在人类胚胎干细胞（hESC）中为Wnt通路动力学创建一种新的报告基因， 和人类癌细胞，时间分辨率为分钟。使用这个工具， 我们发现Wnt配体的刺激产生信号传导的瞬时定位， 效应β-连环蛋白进入细胞核。这是令人惊讶的，因为以前的工作已经表明，我们 我已经证实了我们的记者，总β-连环蛋白水平稳定升高，Wnt 发信号。在这里，我们建议使用自动流体控制的组合来操纵 配体动力学，Wnt途径和细胞命运报告基因的定量测量，以及 数学建模来剖析配体输入、信号转导和信号转导之间的关系。 β-连环蛋白，以及由此产生的细胞命运决定。我们将研究内源性Wnt的作用 使用新的hESC培养平台在人外胚层图案化中的信号传导。在这个系统中， 细胞使用微图案化技术以受控的几何形状生长，分化为 外胚层命运，然后用配体BMP 4处理。我们已经表明，BMP 4的反应， 导致靠近殖民地边界的表皮命运，并且它诱导继发性内源性 Wnt信号导致殖民地内特定半径的神经嵴分化。细胞 在殖民地的中心不接收这些信号，并采取神经的命运。我们将联合收割机 该测定用活细胞测量Wnt和BMP途径活性和细胞命运标志物 为了理解所提供的BMP信号和所诱导的Wnt信号之间的相互作用 创造了外胚层细胞命运的自组织模式。该项目将提供必要的 关于Wnt通路动力学的信息，这对于了解其在胚胎发育中的作用至关重要。 发展，利用它在定向分化协议，并规避它的情况下， 其中Wnt信号传导驱动癌症的生长。此外，了解Wnt在 体外外胚层图案化分析将揭示在细胞中产生自组织组织的原理， 体外，再生医学的中心目标。

Project summary The Wnt pathway plays a crucial role in embryonic development and disease. During early development, Wnt signaling is required for gastrulation and mesendoderm differentiation, and, slightly later, for the differentiation of neural crest cells within the ectodermal layer. While the detailed biochemistry of signal transduction has been studied extensively, much less is known about the dynamics of pathway activity. We have used CRISPR-Cas9 genome engineering to create a novel reporter for Wnt pathway dynamics in both human embryonic stem cells (hESCs) and human cancer cells with temporal resolution on the time scale of minutes. Using this tool, we have found that stimulation by Wnt ligands yields transient localization of the signaling effector β-catenin to the cell nucleus. This was surprising as previous work has shown, and we have confirmed with our reporter, that total β-catenin levels are stably elevated by Wnt signaling. Here, we propose to use a combination of automated fluidic control for manipulating ligand dynamics, quantitative measurements with Wnt pathway and cell fate reporters, and mathematical modeling to dissect the relationship between ligand inputs, signal transduction by β-catenin, and the resulting cell fate decisions. We will examine the role of endogenous Wnt signaling in human ectodermal patterning using a novel hESC culture platform. In this system, cells are grown in controlled geometries using micropatterning technology, differentiated to ectodermal fates, and then treated with the ligand BMP4. We have shown that BMP4 response leads to epidermal fates near the colony border, and that it induces secondary, endogenous Wnt signals that lead to neural crest differentiation at a particular radius within the colony. Cells at the center of colony do not receive these signals, and adopt a neural fate. We will combine this assay with live cell measurements of Wnt and BMP pathway activities and cell fate markers to understand how the interplay between the supplied BMP signal and the induced Wnt signal creates self-organized patterns of ectodermal cell fates. This project will provide essential information on Wnt pathway dynamics, which is crucial for understanding its role in embryonic development, harnessing it in directed differentiation protocols, and circumventing it in cases where Wnt signaling drives the growth of cancers. Further, understanding the role of Wnt in the in vitro ectodermal patterning assay will reveal principles of generating self-organized tissues in vitro, a central goal in regenerative medicine.