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

项目总结 Wnt通路在胚胎发育和疾病中起着至关重要的作用。在早期 在发育过程中，原肠形成和中胚层分化需要Wnt信号。 稍晚一些，用于外胚层内神经脊细胞的分化。而当 信号转导的详细生物化学已被广泛研究，但知之甚少。 关于通路活动的动力学。我们已经使用CRISPR-Cas9基因组工程来 在两种人胚胎干细胞(HESCs)中创建Wnt途径动力学的新报告 和人类癌细胞的时间分辨率在几分钟的时间尺度上。使用这个工具， 我们发现，Wnt配体的刺激产生了信号的瞬时定位 效应器β-连环蛋白进入细胞核。正如之前的工作所表明的那样，这是令人惊讶的，我们 已向本报记者证实，WNT可稳定提高总β-连环蛋白水平 发信号。在这里，我们建议使用自动射流控制的组合来进行操作 配基动力学，用Wnt途径和细胞命运报告进行定量测量，以及 用数学模型分析配基输入、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 使用一种新的hESC培养平台在人类外胚层模式中发出信号。在这个系统中， 使用微图案化技术在受控几何形状中生长细胞，分化为 外胚层命运，然后用BMP4配体处理。我们已经证明了BMP4反应 导致接近菌落边缘的表皮命运，并诱导内源性的次生 在群体内特定半径处导致神经脊分化的WNT信号。单元格 在蜂群的中心没有接收到这些信号，并采取了神经命运。我们将联合起来 用活细胞测量Wnt和BMP途径的活性和细胞命运标志 为了了解提供的BMP信号和诱导的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途径动力学的信息，这对于理解其在胚胎中的作用至关重要 开发，在定向区分协议中利用它，并在案例中绕过它 其中Wnt信号驱动癌症的生长。此外，了解WNT在 体外外胚层图案化实验将揭示自组织形成的原理 体外培养，这是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.