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

Wnt 通路动力学及其在生成自组织外胚层模式中的作用

• 批准号: 10078612
• 负责人: Aryeh Warmflash
• 金额: $ 32.28万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078612
• 关键词: Address; Adopted; Agonist; BMP4; Biochemistry; Biological Assay; CRISPR/Cas technology; Catalogs; Cell Culture System; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cells; Clinic; Data; Development; Disease; Ectoderm; Ectoderm Cell; Embryonic Development; Engineering; Eye; Gene Expression; Genes; Genome engineering; Geometry; Goals; Growth; Human; Image; In Vitro; Individual; Knowledge; Lead; Ligands; Link; Liquid substance; Malignant Neoplasms; Measurement; Molecular; Mutate; Neural Crest; Neural Crest Cell; Output; Pathway interactions; Pattern; Physiologic pulse; Play; Population; Protocols documentation; Radial; Recording of previous events; Regenerative Medicine; Reporter; Resolution; Role; Signal Induction; Signal Transduction; System; Technology; Testing; Time; Tissues; WNT Signaling Pathway; Wnt proteins; Work; beta catenin; cancer cell; cell motility; design; differentiation protocol; experimental study; gastrulation; human embryonic stem cell; inhibitor/antagonist; loss of function; mathematical model; novel; paracrine; relating to nervous system; response; small molecule; stem cells; temporal measurement; tool

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.

项目概要 Wnt 通路在胚胎发育和疾病中起着至关重要的作用。早期期间 原肠胚形成和中内胚层分化需要 Wnt 信号传导，并且， 稍后，用于外胚层内神经嵴细胞的分化。虽然 信号转导的详细生物化学已被广泛研究，但知之甚少 关于通路活动的动态。我们使用 CRISPR-Cas9 基因组工程 为人类胚胎干细胞 (hESC) 中的 Wnt 通路动态创建新型报告基因 以及具有分钟时间尺度的时间分辨率的人类癌细胞。使用这个工具， 我们发现 Wnt 配体的刺激会产生信号传导的瞬时定位 效应子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 可稳定升高总 β-catenin 水平 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 通路和细胞命运报告基因的定量测量，以及 数学模型来剖析配体输入、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 使用新型 hESC 培养平台进行人类外胚层模式信号传导。在这个系统中， 使用微图案化技术，细胞以受控的几何形状生长，可分化为 外胚层命运，然后用配体 BMP4 处理。我们已经证明 BMP4 响应 导致集落边界附近的表皮命运，并诱导继发性、内源性 Wnt 信号导致集落内特定半径处的神经嵴分化。细胞 群体中心不接收这些信号，并采取神经命运。我们将结合 该测定采用活细胞测量 Wnt 和 BMP 通路活性以及细胞命运标记物 了解提供的 BMP 信号和感应的 Wnt 信号之间如何相互作用 创建外胚层细胞命运的自组织模式。该项目将提供必要的 有关 Wnt 通路动态的信息，这对于理解其在胚胎中的作用至关重要 开发，在定向分化协议中利用它，并在案例中规避它 其中 Wnt 信号传导驱动癌症的生长。进一步了解 Wnt 在 体外外胚层模式化测定将揭示在中产生自组织组织的原理 体外，再生医学的中心目标。